WO1994014973A1 - Nouveaux inhibiteurs de neutrophiles - Google Patents

Nouveaux inhibiteurs de neutrophiles Download PDF

Info

Publication number
WO1994014973A1
WO1994014973A1 PCT/US1993/012626 US9312626W WO9414973A1 WO 1994014973 A1 WO1994014973 A1 WO 1994014973A1 US 9312626 W US9312626 W US 9312626W WO 9414973 A1 WO9414973 A1 WO 9414973A1
Authority
WO
WIPO (PCT)
Prior art keywords
inhibitory factor
neutrophil
neutrophil inhibitory
nif
activity
Prior art date
Application number
PCT/US1993/012626
Other languages
English (en)
Inventor
Matthew Moyle
David Lee Foster
George Phillip Vlasuk
Original Assignee
Corvas International, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/060,433 external-priority patent/US6756211B1/en
Priority claimed from US08/151,064 external-priority patent/US6962795B1/en
Application filed by Corvas International, Inc. filed Critical Corvas International, Inc.
Priority to EP94907114A priority Critical patent/EP0682714A4/fr
Priority to JP6515483A priority patent/JPH08505055A/ja
Priority to AU60805/94A priority patent/AU694103B2/en
Publication of WO1994014973A1 publication Critical patent/WO1994014973A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F02—COMBUSTION ENGINES; HOT-GAS OR COMBUSTION-PRODUCT ENGINE PLANTS
    • F02B—INTERNAL-COMBUSTION PISTON ENGINES; COMBUSTION ENGINES IN GENERAL
    • F02B75/00—Other engines
    • F02B75/02—Engines characterised by their cycles, e.g. six-stroke
    • F02B2075/022—Engines characterised by their cycles, e.g. six-stroke having less than six strokes per cycle
    • F02B2075/027—Engines characterised by their cycles, e.g. six-stroke having less than six strokes per cycle four

Definitions

  • This invention relates to factors which interact with CDllb/CD18 ingegrin complex or the I-domain portion of CDllb/CD18 integrin complex and inhibit leukocyte activity. These factors inhibit neutrophil activity, including inhibition of neutrophil activation and adhesion of neutrophils to vascular endothelial cells. These factors also inhibit eosinophil activity, including inhibition of eosinophil adhesion to vascular endothelial cells.
  • Leukocytes are a class of cells comprised of lymphocytes, monocytes and granulocytes.
  • the lymphocytes include within their class, T-cells (as helper T-cells and cytotoxic or suppressor T-cell) , B- cells (as circulating B-cells and plasma cells) , third population or natural killer (NK) cells and antigen- presenting cells.
  • Monocytes include within their class, circulating blood monocytes, Kupffer cells, intraglomerular mesangial cells, alveolar macrophages, serosal macrophages, microglia, spleen sinus macrophages and lymph node sinus macrophages.
  • Granulocytes include within their class, neutrophils, eosinophils, basophils, mast cells, (as mucosa-associated mast cells and connective tissue mast cells) .
  • Neutrophils are an essential component of the host defense system against microbial invasion.
  • neutrophils In response to soluble inflammatory mediators released by cells at the site of injury, neutrophils emigrate into tissue from the bloodstream by crossing the blood vessel wall.
  • activated neutrophils kill foreign cells by phagocytosis and by the release of cytotoxic compounds, such as oxidants, proteases and cytokines.
  • cytotoxic compounds such as oxidants, proteases and cytokines.
  • neutrophils themselves can promote tissue damage.
  • neutrophils can cause significant tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue.
  • neutrophils that stick to the capillary wall or clump in venules may produce tissue damage by ischemia.
  • ARDS adult respiratory distress syndrome
  • ischemia-reperfusion injury following myocardial infarction, shock, stroke, and organ transplantation
  • acute and chronic allograft rejection vasculitis
  • sepsis sepsis
  • rheumatoid arthritis rheumatoid arthritis
  • inflammatory skin diseases Harlan et al., 1990 Immunol. Rev. 114. 5
  • Neutrophil adhesion at the site of inflammation is believed to involve at least two discrete cell-cell interactive events. Initially, vascular endotheliu adjacent to inflamed tissue becomes sticl for neutrophils; neutrophils interact with the endothelium via low affinity adhesive mechanisms in a process known as "rolling". In the second adhesive step, rolling neutrophils bind more tightly to vascular endothelial * cells and migrate from the blood vessel into the tissue. jjt Neutrophil rolling along affected vascular segments
  • L-selectin L-selectin
  • E-selectin endothelial leukocyte adhesion molecule-l or ELAM-1
  • P-selectin granule membrane protein-140, GMP-140, platelet activation-dependent granule-external membrane protein, PADGEM or CD62
  • the counter-receptor for E-selectin is reported to be the sialylated Lewis X antigen (sialyl-Lewis x ) that is present on cell-surface glycoproteins (Phillips et al., 1990 Science 250, 1130; Walz et al., 1990 Science 250, 25 1132; Tiemeyer et al., 1991 Proc. Natl. Acad. Sci. (USA) 88, 1138; Lowe et al. , 1990 Cell j52, 475). Receptors for the other selections are also thought to be carbohydrate in nature but remain to be elucidated.
  • Integrins comprise a broad range of evolutionarily conserved heterodimeric tra smembrane glycoprotein complexes that are present on virtually all 35 cell types.
  • ⁇ 2 leukocyte-specific CD18 family of integrins, which include CDlla/CD18 (LFA-1) and CDllb/CD18 (Mac-1, Mo-1 or CR3) have been reported to mediate neutrophil adhesion to the endothelium (See
  • Endothelial cell counter-receptors for these integrins are the intercellular cell adhesion molecules ICAM-1 and ICAM-2 for CDlla/CD18 and ICAM-1 for cmi b/CD i ⁇ . respectively (Rothlein et al. , 1986 J. Immunol. 137. 1270; Staunton et al., 1988 Cell .52., 925; Staunton et al., 1989 Nature 339, 61) .
  • the ICAMs are monomeric transmembrane proteins that are members of the immunoglobulin superfamily.
  • the CDllb/CD18 integrin is expressed on a variety of leukocytes, including monocytes, macrophages, granulocytes, large granular lymphocytes (NK cells) , and immature and CD5" " B cells (Kishimoto, T.K. , Larson, R.S., Corbi, A.L. , Dustin, M.L. , Staunton, D. ' E. , and Spriger, T.A. (1989) Adv. in Immunol. 46,149-182).
  • CDllb/CD18 has been implicated in a variety of leukocyte functions including adhesion of neutrophils to endothelial cells (Prieto, J., Beatty, P.G.
  • This integrin may play a roll in neutrophil and monocye phagocytosis of opsonized (ie C3bi-coated) targets (Beller, D.I., Springer, T.A. , and Schreiber, R.D. (1982) J.Exp. Med. 156,1000-1009). It has also been reported that CDllb/CD18 contributes to elevated natural killer activity against C3bi-coated target cells (Ramos, O.F., Kai, C. , Yefenof, E. , and Klein, E. (1988) J. Immunol. 140,1239-1243).
  • Endothelial cell agonists which are believed to include small regulatory proteins such as tumor necrosis factor (TNF ⁇ ) and interleukin-l ⁇ (IL-l ⁇ ) , are released by cells at the site of injury. Activation of endothelial cells has been reported to result in the increased surface expression of ICAM-1 (Staunton et al., 1988 Cell 52. 925) and ELAM-1 (Bevilacgua et al., 1987 Proc. Natl. Acad. Sci. (USA) .84., 9238) . Raised levels of expression of these adhesive molecules on the surface of activated endothelial cells is believed to lead to the observed increased adhesivity of neutrophils for the vascular endothelium near sites of injury.
  • TNF ⁇ tumor necrosis factor
  • IL-l ⁇ interleukin-l ⁇
  • Activation of the neutrophil results in profound changes to its physiological state, including shape change, ability to phagocytose foreign bodies and release of cytotoxic substances from intracellular granules. Moreover, activation is believed to greatly increase the affinity of adhesive contacts between neutrophils and the vascular endothelium, perhaps through a conformational change in the CDllb/CD18 integrin complex on the neutrophil surface (Vedder and Harlan, 1988 J. Clin. Invest. 1, 676; Buyon et al., 1988 J. Immunol. 140. 3156) . Factors that have been reported to induce neutrophil activation include IL-l ⁇ ,
  • GM-CSF GM-CSF
  • G-CSF MIP-1
  • IL-8 interleukin-8
  • GM-CSF granulocyte/monocyte-colony stimulating factor
  • G-CSF granulocyte-colony stimulating factor
  • ⁇ TNF ⁇ the complement fragment C5a
  • the microbe-derived peptide formyl-Met-Leu-Phe and the lipid-like molecules leukotriene B4 (LTB 4 ) and platelet activating factor Fuortes and Nathan, 1992, in Molecular Basis of Oxidative Damage by Leukocytes Eds Jesaiti ⁇ , A.J. and Dratz, E.A. (CRC Press) pp. 81-90).
  • phorbol esters e.g., phorbol 12-myristate 13-acetate; PMA
  • PMA phorbol 12-myristate 13-acetate
  • these agonists are believed to activate neutrophils by binding receptors on their surface.
  • Receptors that are occupied by agonist molecules are believed to initiate within the neutrophil a cascade of events that ultimately will result in the physiological changes that accompany neutrophil activation. This process is known as signal transduction.
  • the lipid-like PMA is proposed to affect neutrophil activation by passing through the plasma membrane at the cell surface and directly interacting with intracellular components (i.e., protein kinase) of the signal transduction machinery.
  • Glucocorticoids have long been recognized for their anti-inflammatory properties. Steroid induced inhibition of neutrophils has been reported for several neutrophil functions, including adherence (Clark et al., 1979 Blood 53., 633-641; MacGregor, 1977 Ann. Intern. Med. 8.6, 35-39) . The mechanisms by which glucocorticoids modulate neutrophil function are not well understood, but they are generally believed to involve the amplification or suppression of new proteins in treated neutrophils that play a key role in the inflammatory process (Knudsen et al., 1987 J. Immunol. 139, 4129) .
  • lipocortin ⁇ a group of proteins known as lipocortin ⁇ , whose expression is induced ⁇ in neutrophils by glucocorticoids, has been associated with anti-inflammatory properties (Flower, 1989 Br. J. Pharmacol. 9_4, 987-1015). Lipocortins may exert anti-neutrophil effects by interacting with sites on the neutrophil surface (Camussi et al., 1990 J. Exp. Med. 171. 913-927) , but there is no evidence to suggest that the lipocortins act by directly blocking adhesive proteins on the neutrophil. Apart from their beneficial anti-inflammatory properties, glucocorticoids have been associated with significant side-effects.
  • a second class of anti-inflammatory compounds which are reported as direct inhibitors of neutrophil adhesion to the vascular endothelium are monoclonal antibodies.
  • Monoclonal antibodies that recognize and block ligand-binding functions of some of these adhesive molecules have been reported to act as j-n vivo inhibitors of neutrophil-mediated inflammation.
  • monoclonal antibodies to the CD18 subunit of the CD18 integrin complexes (i.e., CDlla/CD18, CDllb/CD18 and CDllc/CDl ⁇ ) on the surface of neutrophils have been reported to prevent a variety of neutrophil-mediated tissue injury in animal models, including pulmonary edema induced by reperfusion (Horgan et al, 1990 Am. J. Physiol. 259.
  • Patent No. 4,935,234 (June 19, 1990), Schlossman, S.F. et al., U.S. Patent No. 5,019,648 (May 28, 1991) and Rusche, J.R. et al., International Application No. WO 92/11870 (July 23, 1992).
  • Monclonal antibodies directed to CD18 subunit have been reported by Arfors, K.E., U.S. Patent No. 4,797,277 (January 10, 1989), Wright, S.D. et al., European Patent Application No. 346,078 (December 13, 1989), Law, M. et al., European Patent Application No. 438,312 (July 24, 1991), Law, M. et al., European Patent Application No.
  • Antibodies to other adhesive molecules have also been reported to have anti-inflammatory properties. Monoclonal antibodies that recognize the counter-receptor of CDlla/CD18 and CDllb/CD18, ICAM-1 have been reported to prolong cardiac allograft survival (Flavin et al, 1991 Transplant. Proc. 2_2, 533-534) and prevent chemically induced lung inflammation (Barton et al, 1989 J. Immunol. 143. 1278-1282) . Furthermore, anti-selectin monoclonal antibodies have also been reported as active in animal models of neutrophil-mediated inflammation. Monoclonal antibodies to L-selectin have been reported to prevent neutrophil emigration into inflamed skin (Lewinshon et al., 1987 J.
  • E-selectin monoclonal antibodies (Mulligan et al. , 1991 J. Clin. Invest. 8ji. 1396; Gundel et al., 1991 J. Clin. Invest. , 1407) . While monoclonal antibodies to adhesive proteins have demonstrated the feasibility of using neutrophil adhesion inhibitors as anti-inflammatory agents, their utility as therapeutics requires further evaluation.
  • Soluble adhesive receptors obtained by genetic engineering have been proposed as anti-inflammatory compounds.
  • Soluble receptors, in which the transmembrane and intracellular domains have been deleted by recombinant DNA technology, have been tested as inhibitors of neutrophil adhesion to endothelial cells.
  • the functional use of recombinant soluble adhesive molecules has been reported using CDllb/CD18 (Dana et al., 1991 Proc. Natl. Acad. Sci. (USA) ⁇ 88 . , 3106-3110) and L-selectin (Watson et al., 1991).
  • leumedins a new class of anti-leukocyte compounds collectively termed "leumedins” has been reported. These compounds have been reported to block the recruitment in vivo of T lymphocytes and neutrophils into inflammatory lesions. The mechanism of action of the leumedins is unclear, but there is evidence that they do not function by blocking neutrophil activation (Burch et al., 1991 Proc. Natl. Acad. Sci. (USA) 88, 355) . It remains to be determined if leumedins block neutrophil infiltration by direct interference with adhesive molecules.
  • Trichinella spiralis The nematode, Trichinella spiralis. has been reported to either excrete and/or secrete factors which inhibit chemotaxis and p-nitroblue tetrazolium reduction (i.e., release of oxidative metabolites) but enhance chemokinesis of human neutrophils (Bruschi, F. et al., 1989, Wiadomosci Parazytologiczne, .3j5: 391).
  • eosinophils Another component of the host defence mechanism against invading pathogens are eosinophils. Functionally, eosinophils are similar to neutrophils in that both cell types have the ability to phagocytose and to release compounds that are either directly or indirectly toxic to pathogenic organisms. Eosinophils are distinguished from neutrophils by their morphologic features, constituents, products and associations with specific diseases. Although eosinophils have been reported to be capable of killing bacteria in vitro, this class of leukocyte alone is not believed sufficient to defend against bacterial infections in vivo. Instead, it is thought that eosinophils afford primary defense against large organisms such as helminthic parasites (Butterworth AE, 1984; Adv. Parasitol.
  • eosinophils can play a major role in certain inflammatory diseases. Specifically, substances released from eosinophils that are known collectively as cationic granule proteins, including major basic protein, eosinophil cationic protein and eosinophil-derived neurotoxin, have been implicated in asthma (Gleich GJ and Adolphson, CR, 1986; Adv. Immunol. 29_'.177-253) , inflammatory bowel disease (Hallren, R, 1989; Am. J. Med. 6:56-64) and atopic dermatitis (Tsuda, S, et al, 1992; J. Dermatol.
  • eosinophil products such as superoxide anions, hydroxyl radicals and singlet oxygen may also be involved in damage to host tissue in inflammatory disease states (Petreccia, DC et al, 1987,
  • VLA-4 very late antigen-4, a4bl
  • vascular cell adhesion molecule-1 vascular cell adhesion molecule-1 that is expressed on the surface of endothelial cells
  • IL-1 treatment of the endothelial cell monolayers has been reported to induce an increased adhesiveness for human basophils t eosinophils and neutrophils but treatment of these endothelial cells with an antibody directed to VACM-l was reported to inhibit both basophil and eosinophil adhesion but not neutrophil adhesion. It has also been reported that monoclonal antibodies against VCAM-1 inhibit lymphocyte and monocyte cell adhesion to stimulated endothelium (Carlos et al.
  • a second approach to anti-eosinophil therapy has been the use of compounds that directly inhibit the adhesion of eosinophils to vascular endothelium. It has been reported that in animal models of asthma, monoclonal antibody against ICAM-1 blocks eosinophil infiltration into tissues. Wegner et al. (1990), Science, 247:456-459. ICAM-1 and functional derivatives thereof have been proposed as anti-inflammatory agents. Anderson et al., European Patent Application No. 314,863 (April 29, 1988); Wegner et al., International Application No. WO 90/10453 (September 20, 1990).
  • the present invention describes potent and specific inhibitors of neutrophil and eosinophil activity, in particular the adhesion of these granulocytes to vascular endothelial cells, derived from hookworms (such as Ancylostoma caninum) and related species.
  • the present invention is based on our finding that the Neutrophil Inhibitory Factor of the present invention represents a pioneering step toward the development of a new generation of anti-inflammatory therapeutic products.
  • This discovery will enable therapy for inflammatory disease based entirely on specific inhibition of the inflammatory response.
  • the therapeutic advantages of this novel approach are realized through the specificity of Neutrophil Inhibitory Factor compared to current clinical treatment modalities such as steroids, catecholamines, prostaglandins, and nonsteroidal anti-inflammatory agents.
  • the currently used therapeutic agents demonstrate poor efficacy and multiple adverse reactions due to generalized systemic effects that non-specifically target numerous biological processes in addition to the inflammatory process.
  • the Neutrophil Inhibitory Factor of the present invention is believed to meet this need by providing the potential for a lifesaving therapy which is currently being sought throughout the international medical and pharmaceutical research communities.
  • the present invention is believed to fulfill this need by disclosing a potent and specific inhibitor of neutrophil activity, in particular the adhesion of neutrophils to vascular endothelial cell_ ⁇ ', derived from hookworms (such as Ancylostoma caninum) and related species.
  • the present invention is directed to a neutrophil inhibitory factor ("Neutrophil Inhibitory Factor” or "NIF”) which may be isolated from natural sources or made by recombinant methods.
  • Neutrophil Inhibitory Factor is a protein which is neither an antibody, a member of the integrin or selectin families nor a member of the immunoglobulin superfamily of adhesive proteins and which when isolated from a parasitic worm is a glycoprotein.
  • Recombinant NIF produced by certain expression systems may or may not be glycosylated, or may be glycosylated to a variable degree. However, such NIFs whether glycosylated or not are considered to be within the scope of the present invention.
  • the present invention provides NIFs which contain, as part of their total amino acid sequence, an amino acid sequence selected from the group consisting of
  • X n is lie or Tyr;
  • X 12 is Asp, Lys or Glu;
  • X 13 is Asp or Glu ;
  • X 14 is Gly, Lys or Arg; and
  • X 15 is Glu, Met, Thr or Val ;
  • X 29 -Tyr wherein X 23 is Thr , Ser , Lys or Glu; X u is Thr , Val or lie; X ⁇ is Val, Lys or Thr; X 26 is Arg, Ser or Asp; X 27 is Asn, Gly, Asp or Arg; X 28 is Asn, Ser or Thr; and X 29 is Gly, Glu or Asp; and
  • the present invention provides mutant NIFs wherein certain asparagine residues are replaced with glutamine residues which is believed to result in the reduced glycosylation of these NIFs.
  • the mutant NIFs contain, as part of their total amino acid sequence, an amino acid sequence selected from the group consisting of peptides (a) to (f) hereinabove. Such NIFs exhibit neutrophil inhibitory activity.
  • the present invention provides NIFs which contain, as part of their total amino acid seguence, an amino acid sequence encoded by a nucleic acid sequence which is sufficiently complementary to hybridize to certain nucleic acid probes. Such NIFs exhibit neutrophil inhibitory activity.
  • the present invention includes within its scope these nucleic acid probes.
  • the present invention provides nucleic acid molecules encoding for a NIF and which are isolated as described herein.
  • isolated nucleic acid molecules include expression vectors containing a nucleic acid sequence encoding a NIF.
  • the present invention also includes the host cells transformed by such expression vectors.
  • the present invention provides methods for making biologically active NIFs, wherein such NIFs are expressed and, optionally ⁇ secreted.
  • the present invention also includes the NIFs made by these methods.
  • the present invention provides methods of making NIFs comprising preparing a cDNA library from a source suspected of having a NIF and hybridizing certain oligonucleotide probes of the present invention to the nucleic acid molecules from the source. Such NIFs exhibit neutrophil inhibitory activity.
  • the present invention also includes the NIFs made by these methods.
  • the present invention provides methods of detecting in a sample the presence of a nucleic acid molecule encoding a NIF, which methods comprise the combining the sample thought to contain such nucleic acid molecule with a probe of the present invention and detecting the presence of hybridized probe.
  • the present invention provides monoclonal antibodies which bind to NIF.
  • the present invention also includes the hybridoma cell lines which make such antibodies, a method of purifying NIF using such monoclonal antibodies and method of detecting in a sample the presence of NIF using such antibodies.
  • the present invention provides a method for detecting in a sample NIF mimics which compete with NIFs for binding to the CDllb/CD18 receptor, which method comprises contacting a sample with the CDllb/CD18 receptor. Also provided is a method for detecting in a sample NIF mimics which compete with NIFs for binding to the I-domain portion of the CDllb/CD18 receptor, which method comprises contacting a sample with a recombinant peptide comprising the I- domain of CDllb/CD18 receptor. Such NIF mimics exhibit neutrophil inhibitory activity.
  • the present invention also includes the NIF mimics detected b# ' these methods.
  • the present invention provides a method for detecting in a sample a NIF antagonist which prevents NIF binding to the CDllb/CD18 receptor, which method comprises contacting such sample with the CDllb/CD18 receptor. Also provided is a method for detecting in a sample NIF antagonist which compete with NIFs for binding to the I-domain portion of the
  • CDllb/CD18 receptor which method comprises contacting a sample with a recombinant peptide comprising the I- domain of CDllb/CD18 receptor.
  • NIF antagonists do not exhibit neutrophil inhibitory activity themselves.
  • the present invention also includes the NIF antagonists detected by these methods.
  • the present invention provides methods of using NIF to treat inflammatory conditions, especially to prevent or decrease inflammatory responses, which methods comprise administering to a mammal a therapeutically effective amount of NIF.
  • amino acid refers to the natural L-amino acids.
  • the natural amino acids shall be referred to by their names or may be abbreviated as shown below: L-amino acid Abbreviation Three letter One Letter alanine Ala A arginine Arg R asparagine Asn N aspartic acid Asp D cysteine Cys C glutamine Gin Q glutamic acid Glu E glycine Gly G histidine His H isoleucine lie I leucine Leu L lysine Lys K methionine Met M phenylalanine Phe F proline Pro P serine Ser S threonine Thr T tryptophan Trp W tyrosine Tyr Y valine Val V
  • amino acid residue refers to amino acid residue
  • x is 1, 2 or 3 representing the azetidine- carboxylic acid, proline or pipecolic acid residues, respectively.
  • isoform refers to a family of related proteins from a single organism having homologous sequences of amino acid residues interspersed with variable sequences.
  • nucleic acid refers to polymers of either deoxynucleic acids or ribonucleic*acids, either single-stranded or double-stranded.
  • isolated nucleic acid refers to nucleic acids which are isolated by biochemical or molecular biology techniques such as centrifugation, chromatography, electrophoresis, hybridization and the like.
  • NIF mimic refers to a small molecule, peptide, peptide analog or protein, which competes with NIF for binding to the CDllb/CD18 receptor or the I- domain portion of the CDllb/CD18 recptor.
  • a NIF mimic is also characterized as having neutrophil inhibitory activity, eosinophil inhibitory activity or both such activities.
  • NIF antagonist refers to a small molecule, peptide, peptide analog or protein, which prevents the binding of NIF to the CDllb/CD18 receptor or the I-domain portion of this receptor, and. does not possess any significant neutrophil inhibitory activity.
  • a NIF antagonist prevents binding of NIF to the CDllb/ CD18 receptor or the I-domain portion of the CDllb/CD18 receptor, by binding to a site on NIF which is required for binding to the receptor in effect sterically hindering binding, or alternatively, by binding to a site on NIF which results in a conformational change to the site needed for such binding which change substantially weakens or abolishes binding.
  • Figure 1 depicts a chromatogram of hookworm lysate obtained as described in the Example 2(A) run on the Example 2(B) Concanavalin A Sepharose column.
  • Figure 2 depicts a chromatogram of Concanavalin A-purified hookworm lysate run on the Example 2(C) Superdex 200 column.
  • Figure 3 depicts a chromatogram of ⁇ *the Concanavalin A Sepharose/Superdex purified hookworm lysate run on the Example 2(D) ceramic hydroxyapatite column.
  • Figure 4 depicts a chromatogram from reverse phase HPLC of hookworm lysate isolated by Concanavalin A Sepharose, Superdex 200 and hydroxyapatite chromatography as described in Example 1(E).
  • Figure 5 depicts a gel pattern run using SDS-gel electrophoresis of the HPLC isolate and certain molecular weight standards.
  • Figure 6 depicts laser-desorption time-of-flight mass spectrometry of the purified Neutrophil Inhibitory Factor of the present invention.
  • Figure 7 depicts the amino acid sequence of proteolytic fragments prepared from Neutrophil Inhibitory Factor isolated from canine hookworms.
  • Figure 8 depicts the nucleotide sequence of the coding region of Neutrophil Inhibitory Factor CDNA (clone 1FL) and its predicted amino acid sequences.
  • Figure 9 depicts the alignment of the predicted amino acid sequences of several Neutrophil Inhibitory Factor isoform clones.
  • Figure 10 depicts the anti-inflammatory effect of varied doses of Neutrophil Inhibitory Factor isolated from canine hookworms administered intraperitoneally in an animal model of inflammation.
  • Figure 11 depicts the anti-inflammatory effect of Neutrophil Inhibitory Factor isolated from canine hookworms administered either intraperitoneally or intravenously in an animal model of inflammation.
  • Figure 12 depicts the anti-inflammatory effect of recombinant Neutrophil Inhibitory Factor produced in Pichia pastoris administered in vivo in an animal model of inflammation.
  • Figure 13 depicts the effect of recombinant NIF on the inhibition of eosinophil adherence *fc ' o TNF-stimulated HUVEC monolayers. Data points are means of triple determinations of one experiment.
  • Figure 14 depicts genetic map of the expression vector Pma5-NIl/3.
  • the vector contains the following elements: (i) a ColEl type origin of replication (ORI); (ii) the intercistronic region of filamentous phage fl including the origin of replication (fl ORI) ; (iii) the beta-lactamase gene which confers resistance to ampicillin (bla) ; (iv) the chloramphenicol acetyl transferase gene which contains an amber translational stop codon as the result of a single nucleotide substitution (cat-am) ; (v) the phage lambda P R promoter; (vi) a small leader cistron; (vii) the methionyl-NIF encoding region and (viii) two tandemly arranged copies of the central transcription terminator of phage fd (fdT) .
  • the sequence of the Met-NIF expression cassette (blown-up region) is shown in Figure 15. The pma/c family of vectors have been described. See Stanssens et al., Nucl. Acid
  • Figure 15 depicts the nucleotide base sequence of the two-cistron Met-NIF expression cassette of Pma5-NIl/3.
  • the encoded methionyl-NIF and leader peptide are shown in the one-letter code. The following features are indicated: the -35 and -10 regions of the phage lambda P R promoter, the transcription initiation point, the Shine-Dalgarno elements preceding the leader cistron (SD cro ) and the Met-NIF gene (SD) and some restriction sites.
  • the vector was obtained by ligation of the PCR amplified NIF-1FL coding region to the recipient vector which was cleaved by Ncol, treated with the Klenow fragment of DNA polymerase I and subsequently digested with Hindlll (both ligation points are indicated) .
  • the construction scheme fuses the 5'-end of the NIF coding region to an ATG initiator codon.
  • Figure 16 depicts a comparison of the nucleotide and amino acid sequences of NIF proteins from hookworms.
  • the NIF-lFL nucleotide sequence is numbered; this numbering is also used to refer to positions in other genes.
  • PCR-NIF7 and PCR-NIF20 were recovered by PCR-technology: the (regions matching with the) PCR-primers are italicized.
  • AcaNIF7 and NIF-1FL9 differ at only one position (G to E replacement; nucleotide-substitution located at position 647) . The one remaining uncertainty in these sequences is at position 660 in PCR-NIF20. No poly(A+) tail was found in NIF-1FL sequence.
  • Figure 17 depicts a comparison of the potency of recombinant NIF-1FL with that of the recombinant proteins AcaNIF6 and AcaNIF24.
  • the three purified proteins were tested in both the hydrogen peroxide release assay of Example 1(E) (panel A) and the neutrophil-plastic adhesion assay of Example 1(C) (panel B) .
  • Figure 18 depicts a comparison of recombinant NIF-1FL with recombinant AcaNIF4 in the competition binding assay of Example 1(F).
  • the amount of bound biotinylated NIF-lFL was detected with ExtrAvidin conjugated with alkaline phosphatase.
  • Figure 19 depicts the nucleotide and amino acid sequence of a NIF protein from A. ceylanicum (AceNIF3) .
  • the underlined sequence derives from the EcoRI linker that was added onto the CDNA.
  • the sequence which may function as polyadenylation signal (AAUAAA) is also indicated.
  • the encoded protein contains 10 potential N-glycosylation sites (N-X-T/S) .
  • Figure 20 depicts the binding of phages displaying a NIF protein from A. ceylanicum (AceNIF3) to Mac-l.
  • AceNIF3 A. ceylanicum
  • Wells coated with LM2/Mac-l complex were first incubated with varying amounts of Pichia-produced RNIF1 (or buffer in the control experiment) ; after 30 minutes, 10 10 virions displaying either NIF-IFL or AceNIF3 were added and the incubation continued for another 90 minutes.
  • phages were detected with rabbit anti-phage serum and goat anti-rabbit alkaline phosphatase conjugate.
  • the various components were incubated in PBS containing 1 Mm MgCl 2 , 1 Mm CaCl 2 and 0.1% skim-milk (phage samples contained 1% skim-milk) . Unbound material was removed by washing with PBS containing 1 Mm CaCl 2 , 1 Mm MgCl 2 , 0.1% Tween 20 and 0.02% thimerosal. The relative amount of bound phages was calculated by taking the OD 405nm reading obtained in the control experiment as 100%.
  • Figure 21 depicts the synthesis of functional NIF-IFL by PAN-NIF-1FL in either a phage-attached or 'soluble' form.
  • the PAN-NIF-1FL vector contains the following elements:
  • a NIF-IFL containing gene fusion which is placed under the transcriptional control of the Plac promoter.
  • the gene fusion consists of the pelB secretion signal, the NIF-IFL coding region, and the filamentous phage M13 genelll (gill) .
  • a TAG amber translational stop codon is present between the NIF-IFL and the GUI seguences a TAG amber translational stop codon is present.
  • the Ncol and Notl restriction sites used for the cloning of the NIF-IFL coding region are indicated.
  • the bla gene which confers resistance to lOO ⁇ g/ml ampicillin or carbenicillin (bla/Ap R ) .
  • ColEl-NIF-1FL a ColEl-type plasmid borne origin of replication
  • su + bacteria such as TGI
  • the PAN-NIF-1FL phagemid-vector codes for a NIF-lFL-pglll fusion protein (pgiii; product of GUI) which becomes incorporated into filamentous virions upon infection of TG1[PAN-NIF-1FL] cells with M13-VCS 'helper'-phage (Statagene) .
  • PAN-NIF-1FL directs the synthesis of 'free' (not phage-attached) RNIF1 which binds to the anti-NIF MAb 3D2 and to Mac-l.
  • TGI ⁇ (lac-proAB) , hsd ⁇ 5 (r ⁇ Tn ⁇ ") , thi, supE / F'[traD36, lacl q , lacZ ⁇ M15, proA + B + ].
  • WK6 ⁇ (lac-proAB) , galE, strA / F' [lacl q , lacZ ⁇ M15, proA + B + ] .
  • Figure 22 depicts ELISAs with NIF-lFL-displaying phage.
  • NIF-displaying phages or non-displaying control phages, e.g. M13-VCS; in each experiment about 5xl0 9 phage particles were added
  • Mac-l immunopurified on LM2-Sepharose
  • LM2/Mac-1 LM2/Mac-1
  • LM2 or the non-neutralizing anti-NIF Mab 3D2.
  • non-phage-attached 'soluble' NIF (sNIF) was allowed to react with the immobilized material (1 ⁇ M) 30 minutes prior to addition of the phages.
  • Bound phages were detected with a rabbit anti-M13 serum and an alkaline phosphatase conjugated goat anti-rabbit serum.
  • Figure 23 depicts a comparison of the potency of
  • Figure 24 depicts binding of biotinylated rNIF to lymphocytes, monocytes and granulocytes as described in
  • Figure 25 depicts direct concordance of NIF binding and CDllb/CD18 expression as determined*by dual fluorescence analysis flow cytometry of peripheral lymphocyte populations as described in Example 31. Detailed Description of the Invention 1. Neutrophil Inhibitory Factor.
  • the present invention in its various aspects is directed to Neutrophil Inhibitory Factor ("NIF”), a protein that inhibits neutrophil activity and which is not an antibody, an integrin, a selectin or a member of the immunoglobulin superfamily of adhesive proteins and which, when isolated from a parasitic worm, is a glycoprotein.
  • NIF Neutrophil Inhibitory Factor
  • Recombinant NIFs produced by certain expression systems are not glycosylated. Such non-glycosylated NIFs are considered to be within the scope of the invention.
  • the inhibition of neutrophil activity by the NIFs of the present invention includes but is not limited to inhibition of one or more of the following a ⁇ tivities by neutrophils: release of hydrogen peroxide, release of superoxide anion, release of myeloperoxidase, release of elastase, homotypic neutrophil aggregation, adhesion to plastic surfaces, adhesion to vascular endothelial cells, chemotaxis, transmigration across a monolayer of endothelial cells and phagocytosis.
  • Preferred assays include those where inhibition of neutrophil activity is demonstrated by an in vitro assay which determines adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation or adhesion of neutrophils to plastic surfaces.
  • Preferred NIFs would have an IC 50 of about 500 Nm or less, more preferably less than 100 N , as measured by one of these neutrophil activity assays.
  • An IC 50 is that concentration of a NIF giving 50% inhibition of the measured activity (see Example 1) .
  • the NIFs of the present invention are further characterized as also having the ability* ' to bind to the CDllb/CD18 receptor (see Example 14) .
  • Preferred assays for determining the binding of NIF to CDllb/CD18 is described in Example 1(F).
  • the NIFs of the present invention are further characterized as also having the ability to bind to the I-domain portion of the CDllb/CD18 receptor (see Example 32) .
  • a preferred assay for determining the binding of the NIF to the I-domain portion is described in Example 32.
  • the NIFs of the present invention may be further characterized as having eosinophil inhibitory activity.
  • a preferred assay for determining eosinophil inhibitory activity is the inhibition of eosinophil activity demonstrated by an in vitro assay which determines adhesion of neutrophils to vascular endothelial cells as described in Example 29.
  • a preferred NIF would have an IC j0 of about 500 Nm or less, more preferably less than 100 Nm, as measured by this eosinophil activity assay.
  • compositions enriched for NIF comprising NIF and which are a isolated by chromatographic or molecular biology methods, or a combination of both methods, from a parasitic worm, preferably a nematode.
  • Suitable parasitic worms include those selected from species of the phyla Platyhelminthes, Nematoda, Nematomorpha or Acanthocephala.
  • An especially preferred source is endoparasitic hookworm species, such as those found to infect canines. It is believed that certain isoforms of NIF are produced by canine hookworm Ancylostoma species such as Ancylostoma caninum.
  • Another suitable source is the endoparasitic worm species Toxocara canis.
  • Substantially similar compounds may be isolated from other nematode species, as well as from other endoparasites of other phyla*
  • Preferred sources for NIF include parasites, including parasitic worms, particularly endoparasitic nematodes and especially hookworm species, including Ancylostoma braziliense.
  • Bunostomum phlebotomum Cyclodontostomum purvisi r Necator americanus, Necator arqentinus. Necator suillus, and Uncinaria stenocephala.
  • the enriched compositions may be enriched for NIF in one aspect by chromatographic methods, which methods may include chromatography on Concanavalin A Sepharose ® , hydroxyapatite or an anion exchange column, gel filtration chromatography preferably using Superdex ® 200, C4 reverse phase HPLC, isoelectric focusing or a combination of those methods or equivalent methods used for separating proteins or proteinaceous factors.
  • chromatographic methods which methods may include chromatography on Concanavalin A Sepharose ® , hydroxyapatite or an anion exchange column, gel filtration chromatography preferably using Superdex ® 200, C4 reverse phase HPLC, isoelectric focusing or a combination of those methods or equivalent methods used for separating proteins or proteinaceous factors.
  • Concanavalin A in place of Concanavalin A, other immobilized lectins may be used.
  • Superdex ® 200 In place of Superdex ® 200, other acrylamide- or agarose-based gel filtration media which fractionate proteins in the appropriate molecular weight range may be used; these include those sold under the tradenames, Sephacryl ® and Superose ® (Pharmacia) .
  • the enriched composition is comprised of NIF.
  • the NIF therein is a glycoprotein derived from or isolated from a parasitic worm, preferably a nematode, and more preferably a hookworm species, especially a canine hookworm species or, alternatively, a Toxocara species, or a compound, preferably a protein, which is substantially similar to said glycoprotein. It is believed that certain isoforms of said glycoprotein are produced by the canine hookworm Ancylostoma caninum.
  • the compound exhibits selective neutrophil inhibitory activity similar to that of the glycoprotein, and, preferably has an IC 50 of about 500 ⁇ Nm or less, more preferably less than 100 Nm, as measured by neutrophil activity assays such as those described herein.
  • IC 50 of about 500 ⁇ Nm or less, more preferably less than 100 Nm, as measured by neutrophil activity assays such as those described herein.
  • the NIFs of the present invention comprise a purified glycoprotein.
  • a NIF may be determined to be a glycoprotein by evaluating binding to Concanavalin A Sepharose (see Example 2(B)) and by positive testing as a glycoprotein in GlycoTrackTM diagnostic assay for the presence of carbohydrate groups (see Example 7) .
  • glycoprotein having neutrophil inhibitory activity which was isolated from canine hookworms has the following characteristics: This glycoprotein is acidic and exhibits an isoelectric point of about 4.5 as determined by isoelectric focusing (see Example 3) . It has an observed molecular weight of about 41,000 daltons ( ⁇ 3,000) as determined by laser-desorption _. time-of-flight mass spectrometry (see Example 6) . Its behavior when subjected to SDS-polyacrylamide gel electrophoresis indicated that it contained multiple disulfide bonds, since the reduced glycoprotein migrated on the gel at a significantly higher apparent molecular weight (see Example 5) .
  • the glycoprotein was demonstrated to specifically inhibit neutrophil activity and not to act as a general cytotoxin in another cell adhesion assay (see Example 13) .
  • This glycoprotein was demonstrated to inhibit neutrophil adhesion to vascular endothelial cells and homotypic neutrophil aggregation.
  • One such enriched composition (see Example 2(D)) exhibited an IC 50 of about 10 Nm.
  • An IC 50 is that concentration of inhibitor giving 50% inhibition of the measured activity (see Example 1) .
  • This glycoprotein was demonstrated to inhibit peritoneal inflammatory response when administered intraperitoneally or intravenously in an animal model of acute inflammation (see Example 16) .
  • Example 1(E) neutrophil adhesion/spreading on plastic
  • Example 1(C) neutrophil adhesion/spreading on plastic
  • the Example 2(D) preparation had an IC 50 of about 10 Nm.
  • An enriched composition of the neutrophil function inhibitory factor was shown to have no inhibitory effect on platelet aggregation (see Example 13) .
  • a second glycoprotein having neutrophil inhibitory activity has been isolated from Toxocara canis. This glycoprotein has an observed molecular weight of about 20,000 daltons as determined by molecular sieve chromatography. This glycoprotein was demonstrated to inhibit neutrophil adhesion to vascular endothelial cells and neutrophil adhesion/spreading on plastic.
  • the present invention is directed to methods for making enriched compositions comprising NIF, wherein such compositions are isolated from natural sources which may include but are not limited to the parasitic worms.
  • One preferred embodiment comprises isolating these enriched compositions by chromatographic methods, which methods would include chromatography on Concanavalin A- Sepharose ® , hydroxyapatite or an anion exchange column, gel filtration chromatography preferably using Superdex ® 200, C4 reverse phase HPLC, isoelectric focusing or a combination of those methods or equivalent methods used for separating proteins or proteinaceous factors.
  • chromatographic methods which methods would include chromatography on Concanavalin A- Sepharose ® , hydroxyapatite or an anion exchange column, gel filtration chromatography preferably using Superdex ® 200, C4 reverse phase HPLC, isoelectric focusing or a combination of those methods or equivalent methods used for separating proteins or proteinaceous factors.
  • Concanavalin A- in place of Concanavalin A-, other immobilized lectins may be used.
  • acrylamide- or agarose-based gel filtration media which fractionate proteins in the appropriate molecular weight range may be used; these include those sold under the tradenames, Sephacryl ® and Superose ® (Pharmacia) .
  • Preferred methods of preparing enriched compositions comprising NIF comprise subjecting a lysate from a parasitic worm to the following isolation steps (a) chromatography on Concavalin-A Sepharose ® , and (b) gel filtration on Superdex ® 200, and (c) chromatography on ceramic hydroxyapatite.
  • the enriched composition may be further enriched for NIF subjecting it to the further isolation step of reverse phase high performance liquid chromatography (HPLC) using a C4 column. Examples of methods of preparing the enriched compositions according to the present invention are described in Examples 2 to 5.
  • compositions comprising NIF isolated by chromatographic methods are at least about 50% pure, that is, they contain at least about 50% NIF.
  • the composition is enriched at least about 200-fold.
  • substantially pure Neutrophil Inhibitory Factor is prepared.
  • substantially pure is meant at least about 90 percent pure. More preferably the Neutrophil Inhibitory Factor so prepared is chromatographically pure.
  • the present invention is directed to a NIF which includes an amino acid sequence selected from the group consisting of (a) Arg-X 1 -X 2 -Phe-Leu-X 3 -X 4 -His-Asn-Gly-Tyr-Arg-Ser-
  • X 5 -Leu-Ala-Leu-Gly-His-X 6 -X 7 -Ile, wherein X- is Leu or Arg; X 2 is Gin, Lys or Arg; X 3 is Ala or Arg; X 4 is Leu or Met; X 5 is Lys, Arg, Leu or lie; X 6 is Val or lie; and X 7 is Ser, Gly or Asn; (b) Ala-X 8 -X 9 -Ala-Ser-X 10 -Met-Arg-X ⁇ -Leu-X 12 -Tyr-
  • the NIF may include from 1 to 6 of amino acid sequence (a) to (f) above.
  • the NIF includes all of amino acid sequences (a) to (f) .
  • the listed amino acid sequences appear in the following order in the protein (from amino terminal end to carboxy terminal end) : (a) , (b) , (c) , (d) , (e) , (f) . Additional amino acid residues or peptide sequences may be interspersed between the above sequences or may be located at the amino terminal and/or carboxy terminal end of the protein (for example, see Figures 7 and 8) .
  • NIF amino acid sequences of some of the NIF isoforms containing one or more of (a) , (b) , (c) , (d) , (e) or (f) are shown in Figures 9 for canine hookworm NIF, in Figure 16 for Ancylostoma caninum NIF and in Figure 19 for Ancylostoma ceylanicum NIF.
  • NIFs comprising the amino acid sequence depicted in Figure 8.
  • NIFs which have one or more of amino acid sequences (a) , (b) , (c) , (d) , (e) and (f) and exhibit neutrophil inhibitory activity in at least one of the in vitro assay.
  • Suitable assays include those assays which determine adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces.
  • An IC 50 is that concentration of a NIF giving 50% inhibition of the measured activity (see Example 1) .
  • the NIFs of the present invention are further characterized as also having the ability to bind to the CDllb/CD18 receptor (see Example 14) .
  • Preferred assays for determining the binding of NIF to CDllb/CD18 receptor are described in Example 1(F).
  • the NIFs of the present invention are further characterized as also having the ability to bind to the I-domain portion of the CDllb/CD18 receptor (See Example 32) .
  • a preferred assay for determining the binding of NIF to the I-domain portion is described in Example 32.
  • the NIFs of the present invention are also further characterized as having eosinophil inhibitory activity.
  • a preferred assay for determining eosinophil inhibitory activity is the inhibition of eosinophil activity demonstrated by an in vitro assay which determines adhesion of neutrophils to vascular endothelial cells as described in Example 29.
  • Preferred are NIFs having an IC S0 of about 500 Nm or less, more preferably less than 100 Nm, as measured by this eosinophil activity assay.
  • the present invention is directed to NIFs made by methods comprising hybridizing the nucleic acid molecules from a source suspected to contain a NIF to certain oligonucleotide primers or CDNA made from such primers. Such NIFs exhibit neutrophil inhibitory activity.
  • the present invention is directed to these methods of making NIFs.
  • the present invention is directed to NIFs comprising an amino acid sequence which is encoded by a nucleic acid sequence which is sufficiently complementary to hybridize to a primer derived from the amino acid sequence of a NIF.
  • Preferred in the PCR cloning method are single stranded DNA primers of 20-100 nucleotides derived from the sequence of NIF from Ancylostoma canium.
  • primers having the following characteristics: limited degeneracy; adherence to codon usage preferences of the particular species from which the library is constructed and primers that target sequences which are conserved among the twelve Ancylostoma caninum NIF isoforms.
  • Each PCR reaction utilizes two primers: a 5-primer that corresponds to the sense strand and a 3'-primer that corresponds to the antisense strand of the NIF coding seguence.
  • the primers 5-CTCGAATTCT(GATC)GC(ATC)AT(ATC) (CT)T(GATC)GG(ATC)TGGGC- 3' and
  • nucleotides within enclosing parentheses are redundant in that any one nucleotide may be used at the position enclosed by such parentheses.
  • the nucleic acid sequence of the DNA primers are preferably derived from the sequence of NIF from Ancylostoma canium.
  • NIF of the present invention comprises a glycoprotein which has been isolated in substantially pure form.
  • the protein may ⁇ be analyzed to determine an N-terminal sequence, or fragments of the protein can be produced by enzymatic or other specific digestion procedures and the sequence of the terminal amino acids of those fragments determined.
  • Such amino acid sequences even if only between five and six contiguous amino acids in length, will provide sufficient information to determine potential DNA sequences of a gene encoding this protein.
  • oligonucleotides can be synthesized using reported procedures, and such oligonucleotides can be used to probe a genomic or CDNA library from hookworm (or other source) to isolate the gene or fragments thereof encoding the sequenced protein.
  • hookworm or other source
  • these oligonucleotides can be designed using standard parameters such that the oligonucleotide is chosen to encode the chosen amino acid sequence.
  • each oligonucleotide having the same nucleotide base sequence except at specific bases which are varied to take into account the redundancy of the codons that may code for any particular amino acid. It is of course desirable to select an amino acid sequence which is encoded by as few different oligonucleotides as possible.
  • the various redundant codons may be specifically selected to represent those codons that are most preferred in, for example, hookworm nucleic acid.
  • the isolated pure NIF protein can be used to obtain antibodies using known procedures.
  • Such antibodies may include monoclonal or polyclonal antibodies and can be used to screen bacteriophage lambd-agtll expression libraries containing other source (e.g. , hookworm) DNA. In this manner, any particular clone which includes nucleic acid encoding a NIF can be readily identified using standard procedures.
  • Genomic DNA libraries of a hookworm can be formed using standard procedures to isolate the genomic DNA of the hookworm, fractionating that DNA using either a random procedure, such as sonication, or a specific procedure such as restriction endonuclease digestion and ligation of those fragments into an appropriate vector, such as a bacteriophage lambda, plasmid or cosmid vector.
  • a library can be screened for useful clones by nucleic acid hybridization using the oligonucleotide mixtures described above. More preferably, however, a CDNA library can be constructed by isolation of total hookworm RNA, passage of that RNA over an oligo-dT column to purify the poly(A)-containing RNA (i.e..
  • RNA messenger RNA
  • reverse transcription of such RNA to produce DNA fragments representative of the RNA (i.e.. CDNA) .
  • CDNA fragments can be inserted using standard procedures into any desired vector, for example, an expression vector such as a commercially available ______ coli expression vector such as bacteriophage lambda-gtll (for expression in E___ coli) , or into a plasmid pcDNA-1 which can be expressed in mammalian COS7 cells.
  • an expression vector such as a commercially available ______ coli expression vector such as bacteriophage lambda-gtll (for expression in E___ coli)
  • plasmid pcDNA-1 which can be expressed in mammalian COS7 cells.
  • the biological activity of the protein expressed by individual clones of the plasmid expression library can be readily assayed using the neutrophil inhibitory activity assays described herein or other suitable assays.
  • the antibodies described above can be used to probe for immunoreactive protein expressed from clones in the bacteriophage expression libraries (e.g., lambda-gtll). It is particularly preferred to screen various libraries in sub-pools, for example of 999 clones at a time, to determine which of those sub-pools includes a positive clone.
  • a grid of the 999 colonies can be formed on a 33 x 33 plate and ea ⁇ h of the 33 clones in each row and column in the plate assayed simultaneously (i.e.. in 66 preparations) to identify the desired clone.
  • the desired clone Once the desired clone is isolated, its structure is analyzed by standard procedures, for example, by DNA sequencing to determine whether it encodes the whole of the desired protein. If it does not, that clone can be used to screen further CDNA or genomic libraries for full-length clones, or the DNA can be used to hybrid select RNA present in the hookworm, or other source, and more selective CDNA libraries formed from that RNA using procedures described above.
  • the present invention is directed to NIFs comprising an amino acid sequence which is encoded by a nucleic acid sequence which is sufficiently complementary to hybridize to CDNA probes derived from the amino acid sequence of a NIF.
  • probes having at least about 12 nucleotides which are complementary to a portion of the sequence of Figure 8.
  • oligonucleotide primers can be used in the polymerase chain reaction (PCR) to generate complementary DNA probes. These probes can be used to isolate NIF from other sources or isoforms from a single source. Preferred are animal, fungal, bacterial or viral sources.
  • PCR cloning method single stranded DNA primers of 20-100 nucleotides are derived from the sequence of NIF from Ancylostoma canium.
  • Preferred primers have the following characteristics: limited degeneracy; adherence to codon usage preferences of the particular species from which the library is constructed and primers that target sequences which are conserved among the twelve Ancylostoma NIF isoforms.
  • Each PCR reaction utilizes two primers: a 5-primer that corresponds to the sense strand and a 3 ⁇ -primer that corresponds to the antisense strand of the NIF coding sequence.
  • a 5-primer that corresponds to the sense strand
  • a 3 ⁇ -primer that corresponds to the antisense strand of the NIF coding sequence.
  • the primers 5-CTCGAATTCT(GATC)GC(ATC)AT(ATC) (CT)T(GATC)GG(ATC)TGGGC- 3' and
  • nucleotides within enclosing parentheses are redundant in that any one nucleotide may be used at the position enclosed by such parentheses.
  • Single stranded CDNA template is generated using poly(A) + or total RNA prepared from cells of the tissue or organism to be screened.
  • RNA is primed with either random hexanucleotides or oligo d(T) and extended with reverse transcriptase.
  • This reaction product is amplified using an appropriate DNA polymerase (e.g., Taq polymerase) , with a sense and antisense primer, with an appropriate thermocycler.
  • an appropriate DNA polymerase e.g., Taq polymerase
  • Preferred conditions are: cycles 1-3, denaturation at 94°C for 1 minute, annealing at 37°C for l minute and elongation at 72°C for two minutes. The ramp time between annealing and elongation steps is extended to at least 2 minutes for these cycles; cycles 4-40, denaturation at 94°C for 1 minute, annealing at 45°C for 1 minute and elongation at 72°C for two minutes. In subsequent experiments, annealing temperature is increased until a single product results from amplification with each primer pair.
  • Amplification products from individual amplification reactions are used as hybridization probes to screen genomic DNA or CDNA libraries constructed from the tissue from which PCR was effected. DNA or CDNA from any recombinant plaque or colony that hybridized to these amplification products is selected'' for further analyses. NIF complementary DNAs isolated using the techniques described above are subjected to nucleotide sequence analysis using the procedure of dideoxy sequencing (Sanger et al, 1977, Proc. Natl. Acad. Sci USA 24:5463-5467) .
  • NIF CDNA isolates containing open reading frames are inserted into suitable vectors for protein expression in either bacterial, yeast, insect or mammalian cells.
  • Expression systems comprise vectors designed to secrete recombinant protein (i.e., fusion of CDNA isolate open reading frame with a known secretion signal sequence for that cell type) into the culture medium.
  • Vectors lacking a secretion signal sequence are also used for expression.
  • oligonucleotide primers derived from the peptide sequences of NIF isolated from the hookworm
  • Ancylostoma caninum were used in conjunction with the polymerase chain reaction to amplify NIF CDNA sequences. These NIF sequences were used in turn to probe a hookworm CDNA library. Ten full-length and six partial clone isoforms of NIF were isolated in addition to the protypical NIF-IFL full-length clone. This example illustrates the utility of this technique for isolation of sequences that are structurally related to NIF.
  • DNA sequences which encode NIF from other animal, fungal, bacterial or viral source may be isolated and used to express recombinant NIF.
  • the expression vectors described above, or derivatives thereof, can be used for expression of recombinant protein with biological activity. Such recombinant protein is useful in this invention.
  • NIF NIF of the present invention
  • peptide fragments were produced and their amino acid sequences determined. This experiment is described in Example 9.
  • the amino acid sequences obtained for the proteolytic fragments are set forth in Figure 7.
  • the nucleotide sequence for the CDNA of one of the isolated clones (clone 1FL) is depicted in Figure 8.
  • Deduced partial amino acid sequences for other isolated NIF isoform clones are depicted in Figures 9 and 16.
  • NIFs may be isolated from any source, whether, animal, bacterial, fungal, viral or other source suspected of having a NIF.
  • Such NIFs and nucleic acid sequences encoding them may be isolated by methods such as probing a genomic or CDNA library from the source suspected of having a NIF using oligonucleotide probes sufficiently complementary to a nucleic acid sequence encoding a NIF such as those sequences depicted in Figure 8, and then isolating and expressing those nucleic acid sequences which hybridize to the probes as described herein.
  • Such probes have a sufficient number of nucleotides to des ⁇ ribe a unique sequence.
  • probes will have at least about 12 nucleotides.
  • One preferred group of probes include those of the sequences: 5'-CTCGAATTCT(GATC)GC(ATC)AT(ATC)-(CT)T(GATC)GG(ATC)TGGG C-3' and 5'-CTCGAATTCTT(TC)TC-
  • nucleotides within enclosing parentheses are redundant in that any one nucleotide may be used at the position enclosed by such parentheses.
  • NIF proteins and nucleic acids coding for such proteins may be isolated by probing a sample of nucleic acid from a source suspected of having a NIF with an oligonucleotide probe having at least about 12 nucleotides which is complementary to a nucleic acid sequence known to encode a NIF, such as the sequence depicted in Figure 8 and isolating those nucleic acid sequences, such as a gene, which are sufficiently complementary to the oligonucleotide probe to hybridize thereto.
  • the isolated nucleic acid sequence may then be cloned and expressed using art techniques.
  • the present invention is directed to isolated nucleic acid molecules comprising a nucleic acid sequence encoding the amino acid sequence of NIF.
  • the DNA isolate may also include additional sequences which do not code for portions of the finished protein, such as introns, and/or sequences which code for intervening amino acid residues or peptides in addition to the above peptide sequences.
  • Preferred isolated nucleic acid molecules are those which encode a NIF comprising at least one amino acid sequence selected from the group consisting of
  • X 27 is Asn, Gly, Asp or Arg
  • X 28 is Asn, Ser or Thr
  • X 29 is Gly, Glu or Asp
  • the present invention is directed to methods for making biologically active NIFs, wherein such NIFs are expressed intracellularly and are optionally secreted; expression vectors encoding NIF; and host cells transformed with these expression vectors which express and, optionally, secrete NIF.
  • the CDNA encoding NIF may be inserted into a replicable vector for expression, resulting in the synthesis of biologically active recombinant NIF.
  • Many vectors are available for expression of heterologous proteins and selection of the appropriate vector will depend primarily on the desired properties of the host cell.
  • Each of the available vectors contain various components specific to the host cell to be transformed.
  • the vector components or control elements generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, a promoter, an enhancer element and a transcription termination sequence.
  • the expression vector containing the inhibitor is constructed, a suitable host cell is transfected or transformed with the expression vector, and recombinant NIF is purified either from the host cell itself or the host cell growth medium.
  • the signal sequence may be a-component of the vector, or it may be encoded by the NIF DNA that is inserted into the vector. If the native inhibitory factor is a secreted gene product (i.e., from the hookworm (or other source) cells) , then the native pro-NIF from hookworm DNA may encode a signal sequence at the amino terminus of the polypeptide that is cleaved during post-translational processing of the polypeptide to form the mature NIF.
  • All vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
  • this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
  • origins of replication or autonomously replicating sequences are well known for a variety of bacterial, yeast, insect and mammalian cells.
  • the origin of replication from the plasmid PBR322 is suitable for most for most gram-negative ⁇ bacteria, the 2m plasmid origin is suitable for yeast, the baculovirus origin is suitable for some insect cells (e.g.. Sf9 cells; ATCC# CRL1711) and various viral origins (e.g. , SV40, adenovirus) are useful for cloning vectors in mammalian cells.
  • Expression vectors preferably contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin or methotrexate, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media. Expression vectors contain promoters that are recognized by the host organism.
  • Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 base pairs) that control the transcription and translation of a particular nucleic acid sequence, such as hookworm NIF, to which they are operably linked.
  • promoters recognized by a variety of potential host cells are well known. These promoters are operably linked to DNA encoding the NIF by inserting the latter into the vector in a way such that the 5' terminus of the NIF DNA is in close linear proximity to the promoter.
  • Enhancers are cis-acting elements of DNA, usually about 10-300 base pairs in length, that act on- a promoter to increase its transcription. Enhancers are relatively orientation and position independent. Typically, one will use an enhancer from a eukaryotic cell virus for expression in mammalian cells. Examples include the SV40 enhancer, the cytomegalovirus early promoter enhancer and the adenovirus enhancers.
  • Expression vectors used in eukaryotic (i.e., non-bacterial) host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' end and, occasionally from the 3' untranslated regions of eukaryotic or viral DNAs.
  • Preferred expression vectors of the present invention include but are not limited to PHIL7SP-Nlcl0, PSG5/NIF1FLCR1, Pma5-NIl/3 PAN-NIF-1FL, pYAM7SP-hNIFl/ ⁇ ,Gll-5, pYAM7SP-hNIFl/ ⁇ ,Gll-5, pYAMSP-AcaNIF4, pYAMSP-AcaNIF6, pYAMSP-AcaNIF9, pYAMSP-AcaNIF24 and pAN-AceNIF3, the construction of which is described in the Examples.
  • Suitable host cells for the expression vectors described herein include bacterial, yeast, insect or mammalian cells.
  • Preferred bacteria include E ⁇ coli strains
  • preferred yeast include Saccharomyces cerevisiae and Pichia pastoris.
  • a preferred insect cell line is Sf9 (ATCC# CRL 1711)
  • preferred mammalian cell lines are COS-7 (ATCC# CRL 1651) , CHO dhfr' (ATCC# CRL 9096) , CHO-K1 (ATCC# CCL 61) and HeLa (ATCC# CCL 2) .
  • These examples of host cells are illustrative rather than limiting.
  • the host cell should secrete minimal amounts of proteolytic enzymes.
  • Particularly suitable host cells for the expression of glycosylated NIF are derived from multicellular organisms. Such host cells are capable of complex post-translational processing and glycosylation of expressed proteins.
  • Host cells are transfected and preferably transformed with the above-described expression vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters and selecting transformants.
  • Transfection refers to the taking up of an expression vector by a host cell. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, calcium phosphate coprecipitation, spheroplasting transformation and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell. Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or chromosomal integration.
  • transformation is done using standard techniques appropriate to such cells (e.g., calcium chloride or electroporation for bacterial cells; spheroplasting or electroporation for yeast cells; calcium phosphate or electroporation for insect and mammalian cells) .
  • standard techniques appropriate to such cells (e.g., calcium chloride or electroporation for bacterial cells; spheroplasting or electroporation for yeast cells; calcium phosphate or electroporation for insect and mammalian cells) .
  • the recombinant hookworm NIF preferably is recovered from the culture medium as secreted polypeptide, although it may also be recovered from host cell lysates when expressed intracellularly without a signal or secretory sequence.
  • the expressed hookworm NIF may be purified from culture medium or from cell lysates by a variety of separation techniques including, but not limited to, gel filtration, affinity and ion exchange chromatography, hydroxyapatite chromatography,
  • the present invention is directed to mutant NIFs comprising the amino acid sequence shown in Figure 8, wherein one or more of asparagine residues at positions 10, 18,-87, 110, 130, 197 or 223 is replaced by an glutamine residue.
  • Amino acid sequence variants of NIF may be prepared by introducing nucleotide changes into the DNA which encodes NIF, isolated as described above. Such variants include substitutions of residues within the amino acid sequence of the NIF. Any combination of substitutions can be made to arrive at the final construct, provided that the final construct possesses certain desired characteristics. The desired characteristics include, but are not limited to, an increased potency over the wild-type NIF and alteration of the amount of glycosylation of NIFs.
  • mutagenesis with base-specific chemical mutagens as described in detail by Pine and Huang (1987, Methods Enzymol. 154. 415-430) .
  • Another approach is site-directed mutagenesis, for example, as described in Stanssens et al. (1989), Nucl. Acids Res., 17: 4441-4454.
  • Example 25 describes the substitution of certain asparagine residues in the amino acid sequence of a NIF referred to as IFL (see Figure 8) by site-specific mutagenesis.
  • the asparagine residues at positions 10, 18, 87, 110, 130, 197 and 223 of the amino acid sequence of NIF isoform, IFL, are believed to be sites associated with the potential N-linked glycosylation of this isoform.
  • asparagine residues at positions 10, 18, 87, 110 and 130 of IFL were replaced by glutamine residues to yield a mutant NIF.
  • mutant NIF nuclear factor-binding protein
  • the cDNA encoding this mutant was further mutagenized by the same procedure using other oligonucleotides described to produce a mutant NIF, wherein the asparagine residues at positions 197 and 223 of the amino acid sequence of IFL were also replaced with glutamine residues.
  • the expressed mutant NIFs were found to have neutrophil inhibitory activity.
  • the present invention is directed to peptide fragments having neutrophil inhibitory activity which are prepared by proteolytic or chemical methods starting with the chromatographically pure NIF of the present invention.
  • Active peptide fragments may be generated by using enzymatic or chemical techniques.
  • Proteolytic cleavage can be accomplished by digestion of the inhibitor with one or more of the following enzymes: chymotrypsin, trypsin, leucine aminopeptidase, endoproteinase Glu-C, - endoproteinase Lys-C, endoproteinase Arg-C, or endoproteinase Asp-N (Carrey, E.A. , 1989 Protein Structure. A Practical Approach, pp. 117-143, T.E. Creighton, ed. IRL Press, New York) .
  • Chemical digestion of the inhibitor may be accomplished by cyanogen bromide, hydroxylamine, or 2-nitro-5-thiocyanobenzoate cleavage (Carrey, E.A., 1989, ibid.) .
  • Sugar moieties can be removed from either the peptide fragments or intact neutrophil inhibitory protein enzymatically with one or more of the following enzymes: glycopeptidase F, endoglycosidase H, endoglycosidase F, or endoglycosidase
  • glycosylation of the intact inhibitor may be suppressed by expression of the protein in bacterial cells or by the inclusion of inhibitors of glycosylation in the eukaryotic cell culture growth medium.
  • Inhibitors of glycosylation and their uses are described in the art (e.g., Keesey, J. 1987 Biochemica Information, pp. 135-141, J. Keesey, ed. , Boehringer Mannheim Biochemicals, Indianapolis) . Separation of active fragments from inactive fragments may be accomplished by conventional, low, medium, or high pressure chromatographic techniques known in the art.
  • the present invention is directed to polyclonal and monoclonal antibodies which have the ability to bind to NIFs.
  • any one of a number of conventional techniques which are known in the art can be employed.
  • polyclonal antibodies are synthesized by injecting an animal (for example a rabbit) with one or more NIF of the present invention. After injection, the animal produces antibodies to these NIFs.
  • the antibody concentration or titer
  • antibody-containing blood is then drawn from the animal, antiserum is prepared from the blood, and the compound-specific antibody is isolated from other antibodies in the serum by any one of a number of separation techniques (for example, affinity chromatography) .
  • Monoclonal antibodies may be prepared using the technigues of Kohler and Milstein, Nature 256. 495-497 (1975) as well as other conventional techniques known to those skilled in the art. (See, e.g. , Harlow and Lane, Antibodies. A Laboratory Manual (Cold Spring Harbor Laboratory, 1988) the disclosures of which is incorporated herein by reference) .
  • Preferred monoclonal antibodies include those directed .to the NIF isoform,
  • IFL whose amino acid sequence is depicted in Figure 8 and which are immunoglobulins of the IgG*class.
  • Especially preferred monoclonal antibodies are those which bind to the same epitope on this NIF as is bound by the monoclonal antibody, 3D2.
  • the monoclonal antibody referred to as 3D2 is a preferred embodiment. The preparation of 3D2 is described in Example 26.
  • the present invention is directed to hybridomas which produce such monoclonal antibodies. These hybridomas are produced by conventional techniques such as those described by Harlow and Lane, Id. , the disclosures of which is incorporated herein by reference. The preparation of a preferred hybrido a is described in Example 26.
  • the present invention is directed to methods of affinity purification of NIF from various sources and impure compositions of NIF derived from such sources using an antibody to NIF. Preferred method of isolating NIF would comprise the step of contacting a sample thought to contain a NIF ⁇ with a monoclonal antibody which is capable of binding to said NIF.
  • the monoclonal antibody is either the monoclonal antibody, 3D2, or a monoclonal antibody binding to the same epitope on said NIF as is bound by the monoclonal antibody, 3D2.
  • affinity purification the monoclonal antibody is covalently attached to a chromatographic resin.
  • chromatographic resins include E phaze 611 Biosupport Medium (3M Corp.). Examples 27 and 28 describe preparation of a chromatographic resin coupled with the monoclonal antibody, 3D2, and its use to purify NIF from compositions comprising NIF.
  • the present invention is directed to immunoassays using the antibodies against NIF.
  • an immunoassay format is selected.
  • suitable immunoassays are described by Harlow and Lane, Id. (See especially pages 553 to 612) ⁇ ' the disclosures of which are incorporated herein by reference.
  • Immunoassays utilizing the solid phase method or liquid phase method are well known to one skilled in the art of immunoassays.
  • monoclonal antibodies may be used to assay for drugs, hormones and proteins with such assays being in a solid phase or liquid phase format.
  • the preferred methods of the present invention are solid phase assays.
  • the preferred methods of detecting NIF in a sample comprise contacting a sample thought to contain a NIF with a monoclonal antibody which is capable of binding to such NIF.
  • the monoclonal antibody is immobilized onto a plastic surface such polystyrene, polypropylene, polyethylene, nylon and the like.
  • the plastic surface may be configured in the shape of test tube, microspheres, macroscopic beads, microtiter plates and the like.
  • monoclonal antibodies may be attached to the plastic surface by either covalent coupling or by passive absorption, preferably by passive absorption.
  • Preferred as monoclonal antibodes are those which bind to the same epitope on a NIF as is bound by the monoclonal antibody, 3D2, or the monoclonal antibody,
  • the monoclonal antibody may be contacted simultaneously with sample and a NIF which has been covalently linked to a detectable label.
  • the monoclonal antibody may first be contacted with the sample, then with a NIF which has been covalently linked to a detectable label.
  • Preferred detectable labels are enzymes, fluorescent compounds or radioisotopes.
  • preferred detectable labels are enzymes such as alkaline phosphatase, ⁇ -galactosidase or horseradish peroxidase, or radioisotopes such as iodine-125.
  • the manner of covalently linking such enzymes and radioisotopes to monoclonal antibodies is well known to one skilled in the art of diagnostic assays. 4. Methods of Detecting NIF Mimics and NIF Antagonists.
  • the present invention is directed to a method of detecting in a sample the presence of a NIF mimic which competes with NIF for binding to CDllb/CD18 receptor or the I-domain portion of the CDllb/CD18 receptor.
  • the present invention is directed to a NIF antagonist which prevents NIF from binding to the CDllb/CD18 receptor or the I-domain portion of the CDllb/CD18 receptor.
  • the methods comprise contacting said sample with CDllb/CD18 receptor or the I-domain portion of the CDllb/CD18 receptor.
  • NIF mimics and NIF antagonists include but are not limited to small molecules, peptides, peptide analogs or proteins.
  • NIF mimics or antagonists to be tested are preincubated in solution with neutrophils, or immobilized CDllb/CD18 receptor or a recombinant peptide comprising the I-domain portion of the CDllb/CD18 receptor, and the preincubated solution is then brought into contact with labeled NIF.
  • the effect of test compound on the binding of NIF to neutrophils, immobilized CDllb/CD18 receptor or recombinant peptide comprising the I-domain portion of the CDllb/CD18 receptor is then determined.
  • the assay method uses neutrophils which are free in solution.
  • NIF which has been linked to a detectable label
  • neutrophils and a sample thought to contain a NIF mimic or NIF antagonist are co-incubated in solution for a sufficient time to allow binding to occur. Unbound labeled NIF is removed from bound NIF by methods such as centrifugation, filtration or other suitable methods and bound NIF is determined by means of the detectable label.
  • neutrophils are immobilized on a plastic surface by passive absorption or chemical fixation such as by glutaraldehyde or similar chemicals. Labeled NIF is co-incubated with the immobilized neutrophils and a sample thought to contain a NIF mimic or NIF antagonist. Unbound labeled NIF is removed by washing and bound labeled NIF is then determined by means of the detectable label.
  • CDllb/CD18 receptors from a detergent extract of human leukocytes are captured by anti-CDllb/CD18 monoclonal antibody which is immobilized to a plastic surface.
  • a NIF which is linked to a detectable label or a NIF which can be subsequently linked to a detectable label, and sample thought to contain a NIF mimic or NIF antagonist are co-incubated with the immobilized CDllb/CD18 receptor. After the binding has occurred, unbound NIF JLs removed by washing. Detectable label is then added which links to NIF rendering it detectable (if the NIF was not originally linked to such label) . Bound NIF is then determined by means of the detectable label.
  • Anti-CDllb/CD18 antibody may be coupled to a plastic surface by covalent coupling or passive absorption, though passive absorption is preferred.
  • Preferred plastic surfaces are polystyrene, polypropylene, polyethylene, nylon and the like, though polystyrene is preferred.
  • the plastic surface may be configured in the shape of test tube, microspheres, macroscopic beads, microtiter plates and the like.
  • a preferred anti-CDllb/CD18 antibody is the monoclonal antibody referred as LM2.
  • NIF is linked to a detectable label or is capable of being linked to such label during a step of an assay method of the present invention.
  • NIF is linked to a detectable label by covalent coupling ⁇ sing homobifunctional crosslinking reagents such as glutaraldehyde, disuccinimidyl suberate, dimethyl suberimidate and the like.
  • NIF is made capable of being linked to a detectable label during a step of an assay method of the present invention by first linking biotin to NIF and avidin to the detectable label.
  • Preferred detectable labels include enzymes, fluorophores or radioisotopes.
  • Especially preferred detectable labels include alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase, or iodine-125.
  • a recombinant peptide comprising the I-domain of the CDllb/CD18 receptor which is complexed to a monoclonal antibody that recognizes a portin of this peptide NIF which is linked to a detectable label or a NIF which can be subsequently linked to a detectable label, and sample thought to contain a NIF mimic or NIF antagonist are co- incubated.
  • a .protein A- Sepharose is added to separate bound from unbound NIF. Setectable label is then added which links to NIF rendering it detectable (if the NIF was not originally linked to such label) . Bound NIF is then detrmined by means of the detectable label.
  • a NIF mimic or NIF antagonist may be assayed for neutrophil inhibitory activity.
  • Neutrophil inhibiting activity may be demonstrated by an assay such as those assays which determine adhesion of neutrophils to vascular endothelial cells, release of hydrogen peroxide from neutrophils, homotypic neutrophil aggregation and adhesion of neutrophils to plastic surfaces.
  • NIF mimics are characterized as having an IC 50 for inhibiting neutrophil activity of about 500 nM or less, though an IC J0 is about 100 nM or less is preferred.
  • NIF antagonists are characterized having such an IC 50 of about 1,000 nM to about ImM, although an IC J0 of about 5,000 nM to about 10 mM is preferred.
  • An IC j0 is that concentration of a NIF mimic or NIF antagonist giving 50% inhibition of the measured activity (see Example l) .
  • a NIF mimic or NIF antagonist may be assayed for eosinophil inhibitory activity. Eosinophil inhibiting activity is demonstrated by an assay which determines adhesion of eosinophils to vascular endothelial cells. If assayed, NIF mimics are characterized as having an IC 50 for inhibiting eosinophil activity of about 500 nM or less, though a IC 50 is about 100 nM or less is preferred.
  • NIF antagonists are characterized having such an IC 50 of about 1,000 nM to about 1 mM, though an IC J0 of about 5,000 nM to about 10 mM is preferred.
  • An IC 50 is that concentration of a NIF mimic or NIF antagonist giving 50% inhibition of the measured activity.
  • the present invention is directed to NIF mimics and NIF antagonists discovered by the above-disclosed methods of detection of this section.
  • the present invention is directed to pharmaceutical compositions comprising NIF.
  • compositions may be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions for injectable administration; and the like.
  • the dose and method of administration can be tailored to achieve optimal efficacy but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
  • an amount between 0.01 mg/kg to 100 mg/kg body weight/day is administered dependent upon the potency of the composition used.
  • Preferred embodiments encompass pharmaceutical compositions prepared for storage and subsequent administration which comprise a therapeutically effective amount of NIF or an enriched composition of NIF, as described herein in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences. Mack Publishing Co. (A.R. Gennaro edit. 1985).
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives.
  • antioxidants and suspending agents may be used.
  • the present invention is directed to methods of preventing in a mammal an inflammatory condition characterized by abnormal neutrophil activation or abnormal eosinophil activation comprising administering to said mammal a therapeutically effective amount of a NIF or their pharmaceutical compositions.
  • NIFs or their pharmaceutical compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These compositions can be utilized in vivo, ordinarily in a mammal, preferably in a human, or in vitro.
  • compositions can be administered to the mammal in a variety of ways, including parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms.
  • parenterally intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms.
  • the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the mammalian species treated, the particular composition employed, and the specific use for which these compositions are employed.
  • the determination of effective dosage levels that is the dosage levels necessary to achieve the desired result, will be within the ambit of one skilled in the art.
  • applications of compositions are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved.
  • the dosage for a NIF or its pharmaceutical compositions can range broadly depending upon the desired effects and the therapeutic indication. Typically, suitable dosages will be between about 0.01 mg and 100 mg/kg, preferably between about 0.01 and 10 mg/kg, body weight. Administration is preferably parenteral, such as intravenous on a daily or as-needed basis.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride or the like.
  • the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like.
  • absorption enhancing preparations e.g., liposomes may be utilized.
  • the present invention is directed to inhibitors of the leukocytes, and in a preferred aspect, to those leukocytes which have or express CDllb/CD18 receptors.
  • Leukocytes having or expressing the CDllb/CD18 integrin receptor are well known and include the monocytes, macrophages, granulocytes, large granular lymphocytes (NK cells) , and immature and CD5 + B cells (Kishimoto, T.K. , Larson, R.S., Corbi, A.L., Dustin, M.L., Staunton, D.E., and Spriger, T.A. (1989) Adv. in Immunol. 46,149-182).
  • CDllb/CD18 has been implicated in a variety of leukocyte functions including adhesion of neutrophils to endothelial cells (Prieto, J. , Beatty, P.G., Clark, E.A., and Patarroyo, M. (1988) Immunology 63, 631-637; Wallis, W.J., Hickstein, D.D., Schwartz, B.R., June, C.H., Ochs, H.D., Beatty, P.G., Klebanoff, S.J., and Harlan, J.M. (1986) Blood 67, 1007-1013; Smith, C.W. , Marlin, S.D., Rothlein, R.
  • This integrin may play a roll in neutrophil and monocye phagocytosis of opsonized (ie C3bi-coated) targets (Beller, D.I., Springer, T.A. , and Schreiber, R.D. (1982) J.Exp. Med. 156,1000-1009). It has also been reported that CDllb/CD18 contributes to elevated natural killer activity against C3bi-coated target cells (Ramos, O.F., Kai, C, Yefenof, E., and Klein, E. (1988) J. Immunol. 140,1239-1243).
  • the NIFs of the present invention have potent neutrophil inhibitory activity and, thus, may be used as an inhibitors of neutrophil activity, including neutrophil activation in vitro, as well as for preventing or treating in a mammal inflammatory conditions characterized by abnormal neutrophil activation.
  • NIF will be useful in the treatment of inflammation in which the abnormal activation of neutrophils plays a significant role. While applicants do not wish to be bound to any theory or mode of activity, it is believed that this compound will interfere with the inflammatory response which is set into action by neutrophil-endothelial cell interactions. Thus, where adhesion of neutrophils to the endothelium is prevented, the neutrophils will be unable to transmigrate to tissue to elicit a proinflammatory response with consequent tissue damage. Inhibition of neutrophil-neutrophil adhesion and/or aggregation by these NIFs should also prevent microvascular occlusion.
  • these NIFs will be useful in treating a variety of clinical disorders, including shock, stroke, acute and chronic allograft rejection, vasculitis, autoimmune diabetes, rheumatoid arthritis, inflammatory skin diseases, inflammatory bowel disease, adult respiratory distress syndrome (ARDS) , ischemia-reperfusion injury following myocardial infarction, in which neutrophil infiltration and activation has been implicated and acute inflammation caused by bacterial infection, such as sepsis or bacterial meningitis.
  • shock, stroke, acute and chronic allograft rejection vasculitis
  • autoimmune diabetes rheumatoid arthritis
  • inflammatory skin diseases inflammatory bowel disease
  • ARDS adult respiratory distress syndrome
  • ischemia-reperfusion injury following myocardial infarction in which neutrophil infiltration and activation has been implicated and acute inflammation caused by bacterial infection, such as sepsis or bacterial meningitis.
  • NIF neutrophil activity
  • the specific activities of NIFs in carrying out these related functions makes it particularly useful as therapeutic and/or diagnostic agents.
  • Antibodies both monoclonal and polyclonal, directed to NIF are useful for diagnostic purposes and for the identification of concentration levels of the subject peptides in various biological fluids. Immunoassay utilizing these antibodies may be used as a diagnostic test, such as to detect infection of a mammalian host by a parasitic worm or to detect NIF from a parasitic worm in a tissue of the mammalian host.
  • immunoassays may be used in the detection and isolation of NIF from tissue homogenates, cloned cells and the like.
  • NIFs can be used in a test method to screen other compounds to detect NIF mimics or to detect NIF antagonists for their ability to affect NIF binding to the CDllb/CD18 receptor.
  • NIF with suitable adjuvants can be used as a vaccine against parasitic worm infections in mammals.
  • Immunization with NIF vaccine may be used in both the prophylaxis and therapy of parasitic infections.
  • NIF fragments and synthetic polypeptides having the amino acid sequence of NIF may also be used as vaccines.
  • Disease conditions caused by parasitic worms may be treated by - administering to an animal infested with these parasites substances which antagonize NIF (such as NIF antagonists) .
  • Compounds may be screened for their anti-NIF effect according to the screening method described herein above. Examples of such antihelminic agents include antibodies to NIF, both naturally occurring antibodies isolated from serum and polyclonal and monoclonal antibodies described above. Chemically synthesized compounds which act as inhibitors of NIF also are suitable antihelminic agents.
  • the Neutrophil Inhibitory Factor of the present invention demonstrated activity in inhibiting neutrophil function as measured by neutrophil-HUVEC and neutrophil-plastic adhesion assays, homotypic neutrophil aggregation assay and hydrogen peroxide release assay.
  • This inhibitory factor was isolated from hookworm tissue lysates as an enriched composition by a variety of methods including gel filtration chromatography, chromatography on hydroxyapatite and concanavalin A sepharose, C4 reverse-phase HPLC, Mono-Q ion exchange chromatography and preparative isoelectric focusing. The isolated factor appears to inhibit neutrophil adhesion to endothelial cell monolayers by inhibiting neutrophil activation.
  • HUVEC Primary human umbilical vein endothelial cells
  • Clonetics San Diego, CA
  • FBS fetal bovine serum
  • HUVEC were passaged twice and used to seed fibronectin-coated 96 well microtiter plates (Collaborative Research, Bedford, MA) for adhesion assays.
  • protease inhibitors E64, pepstatin A, chymostatin and APMSF were obtained from Calbiochem (La Jolla, CA) .
  • Neutrophils were isolated using Mono-Poly resolving medium (ICN Biomedicals, Costa Mesa, CA) from either heparinized or citrated human blood following the instructions of the manufacturer. Neutrophils were resuspended in HSA buffer (RPMI1640 with 10 mM HEPES pH 7.4, 1.2 mM CaCl, 1.0 mM MgCl, 1% human serum albumin) at a concentration of 6.6xl0 6 cells/ml and used within one hour after isolation.
  • HSA buffer RPMI1640 with 10 mM HEPES pH 7.4, 1.2 mM CaCl, 1.0 mM MgCl, 1% human serum albumin
  • Neutrophils were fluorescently labelled by the following procedure.
  • the cells were washed once in Hank's balanced salt solution (HBSS) and resuspended at lxlO 7 cells/ml in HBSS containing 20 mg/ml calcein (Molecular Probes; Eugene, OR) .
  • the calcein was initially solubilized in 50 ml dry dimethylsulfoxide prior to its addition to the HBSS.
  • Cells were incubated at 37°C with occasional mixing by inversion. After 45 minutes incubation the cells were chilled on ice for 5 minutes and then washed twice with ice-cold HSA buffer. Labelled neutrophils were resuspended in HSA buffer at 1.3xl0 7 cells/ml for use in adhesion assays.
  • the molar protein concentration of purified NIF isoforms and mutants thereof was determined spectrophotometrically at 278 nm thereby using calculated extinction coefficients. The calculation is based on absorbance values of 5600 cm" 1 .mol" 1 for tryptophan and 1420 cm' 1 .mol” 1 for tyrosine residues.
  • Neutrophils were incubated with the HUVEC monolayer for 30 minutes at 37°C to remove non-adherent cells, wells were first filled with 250 ml HSA buffer, sealed with parafilm and then centrifuged inverted for 3 minutes at 75 X g. Inverted plates were then placed on a rocking platform shaker for 5 minutes, after which contents were decanted off and wells were washed twice with 100 ml HSA buffer. Adherent neutrophils were lysed in 100 ml 0.1% (v/v) Triton X-100 (in 50 mM Tris HCl pH 7.4), and agitated for 10 minutes on a plate shaker.
  • Neutrophils (20 ml at 6.6X10 6 cells/ml) were incubated with 5 ml PMA (0.8 mM) for 5 minutes at room temperature in a 0.5 ml polypropylene test tube. Twenty microliters of test fraction, diluted in HSA buffer, was added and the suspension was mixed gently. Aliquots of 10 ml of this suspension were added in triplicate to microtiter wells of 60-well HCA (Terasaki) plates (Nunc, Naperville, IL) . Neutrophils were incubated 5 minutes at 37°C and non-adherent cells were removed by submerging the plate 6 times in HBSS.
  • Adherent neutrophils were quantitated by counting under an inverted light microscope. Binding was quantitated visually. PMA-activated neutrophils spread and adhere tightly to polystyrene plastic. Non-activated neutrophils (i.e., in the absence of PMA) remain round and translucent and do not adhere tightly to plastic. Adherent neutrophils were larger, rhomboid in shape and more opaque, with a granular appearance. In the absence of Neutrophil Inhibitory Factor, greater than 80% of PMA-activated neutrophils rapidly and irreversibly bound plastic, underwent shape change and were not removed by the gentle wash procedure.
  • fractions containing the Ancylostoma Neutrophil Inhibitory Factor exhibited a profound inhibitory effect on plastic binding by activated neutrophils.
  • the hydroxyapatite pool preparation of hookworm Neutrophil Inhibitory Factor inhibited neutrophil adhesion to plastic in this assay with an IC 50 of about 10 nM.
  • Neutrophils 60 ⁇ l at 8xl0 6 cells/ml- in HSA buffer were incubated with 15 ⁇ l PMA (2.4 mM) and 5 ⁇ l CaCl2 0.05 M. After gently mixing 80 ⁇ l of the stimulated cell suspension were added to 96-well high binding polystyrene microtiter plates (Costar) containing 20 ⁇ l of the test sample diluted in HSA buffer. After 45 minutes incubation at 37°C, non-adherent cells were removed by submerging the plates 4 times in PBS. Adherent cells were loaded with dye by adding lOO ⁇ l of
  • Crystal violet indicator (CAS 548-62-9) solution. After 10 min at room temperature plates were rinsed by submerging 4 times in PBS. Lysis of stained adherent cells was done by adding lOO ⁇ l of 1% (v/v) Triton X-100 solution. Absorbance was determined at 605nm with a Thermomax plate reader to quantitate adherent neutrophils.
  • Neutrophil aggregation was performed at 37°C in a Scienco dual channel aggregometer (Morrison, CO) .
  • Neutrophils 190 ml at 6.6X10 6 cells were preincubated with 200 ml test fraction (diluted in HSA Buffer) in a glass cuvette (Scienco) for 2 minutes at room temperature.
  • Ten microliters of PMA were added to initiate aggregation (80 nM final) .
  • the inhibition of neutrophil aggregation was measured at the maximum aggregation response 5 minutes after the addition of PMA.
  • the hydroxyapatite pool preparation of. Neutrophil Inhibitory Factor (see Example 1(D)) inhibited neutrophil adhesion with an IC 50 of about 10 nM.
  • Neutrophils (6.6X10 6 cells/ml) were incubated with test fractions in Release Assay Buffer (HBSS with 25 mM glucose, 10% FBS, 200 mg/ml phenol red, 32 mg/ml horseradish peroxidase) for 5 minutes at 37°C
  • Incubation vessels consisted of 1.5 ml plastic test tubes that were precoated with HBSS containing 50% FBS at 37°C for 60 minutes; coated tubes were washed twice with 0.15 M NaCl before use.
  • FM1P (Sigma; St. Louis,
  • the hydroxyapatite pool preparation of hookworm Neutrophil Inhibitory Factor inhibited hydrogen peroxide release from neutrophils with an IC 50 of about 10 nM.
  • Microtiter polystyrene plates (Costar - high binding; 96 well) were coated with the CDllb/CD18 binding, non-neutralizing mouse monoclonal antibody LM2 (50 ⁇ l of the purified LM2 MAb at a concentration of 10 ⁇ g/ml in 0.1 M NaHC0 3 ; pH 9.5; LM2 ATCC hybridoma #HB204) by overnight incubation at 4°C After removal of the antibody, the wells were blocked with 200 ⁇ l PBS containing 1% (w/v) Skim-milk (Difco Laboratories) at room temperature. After 2 hours the blocking solution was removed and wells were washed 3 times with 200 ⁇ l of PBS.
  • LM2 non-neutralizing mouse monoclonal antibody
  • the immobilized LM2 monoclonal antibody was used to immuno-capture the detergent solubilized CDllb/CD18 receptor as follows. Neutrophils were isolated using PolymorphprepTM (Nycomed) from either citrated whole blood or from buffy-coat following the instructions of the manufacturer. The neutrophil pellet from a 50 ml buffy coat was resuspended in 40 ml RPMI and phorbol myristate acetate (PMA) to a final concentration of 0.8 ⁇ M was added. The suspension was gently rotated at room temperature for 20 minutes.
  • PolymorphprepTM PolymorphprepTM
  • PMA phorbol myristate acetate
  • the cells were spun down and resuspended in 5 ml 0.02 M Tris-HCl, 0.15 M NaCl, 0.001 M MgCl 2 , 0.001 M CaCl 2 . Cells were lysed by adding 5 ml buffer containing 2% Triton ⁇ -100 (Bio-Rad
  • NIF NIF-IFL and isoforms or engineered variants
  • NIF-IFL NIF-IFL, other NIF proteins, and NIF mutants
  • binding of NIF-IFL to the LM2/CDllb/CD18 complex was measured by competition with biotinylated recombinant NIF-IFL produced in Pichia (see Example 12) .
  • Samples containing a constant amount of biotinylated recombinant NIF-IFL and varying amounts of unlabeled recombinant NIF-IFL were prepared in PBS containing
  • Frozen canine hookworms were obtained from Antibody Systems (Bedford, TX) . Hookworms were stored at -70°C until used for homogenate.
  • Hookworms were homogenized on ice* ' in homogenization buffer [0.02M Tris-HCl pH 7.4, 0.05 M NaCl, 0.001 M MgCl 2 , 0.001 M CaCl 2 , 1.0 x 10" 5 M dithiothreitol, 1.0 x 10 "5 M E-64 Protease Inhibitor (CAS 66701-25-5), 1.0 x lO ⁇ M pepstatin A (isovaleryl-Val-Val-4-amino-3-hydroxy- 6-methyl-heptanoyl-Ala-4-amino-3-hydroxy-6-methyl- heptanoic acid, CAS 26305-03-3), 1.0 x 10" 5 M chymostatin (CAS 9076-44-2), 2.0 x 10" 5 M APMSF (amidinophenyl- methylsulfonyl fluoride-HCl) , 5% (v/v) glycerol] using a Tekmar Tissuemizer homogen
  • protease inhibitors E64, pepstatin A, chymostatin, and APMSF were obtained from Calbiochem (La Jolla, CA) .
  • Approximately 3-6 ml of homogenization buffer was used to homogenize each gram of frozen worms (approximately 500 worms) .
  • Insoluble material was pelleted by two sequential centrifugation steps: 40,000 X g,, ⁇ at 4°C for 20 minutes followed by 105,000 x g nuu -at 4°C for 40 minutes.
  • the supernatant solution was clarified by passage through a 0.2 mm cellulose acetate filter (CoStar) .
  • Hookworm lysate (79 ml) was adsorbed to 16 ml of Concanavalin A Sepharose (Pharmacia) pre-equilibrated with Con A buffer [0.02 M Tris-HCl, pH 7.4, 1 M NaCl,
  • Figure 1 depicts Concanavalin A Sepharose chromatography of the hookworm lysate performed as described above. Absorbance at 280 nm was plotted as a function of time.
  • Active fractions eluted from immobilized Concanavalin A (see step (B) above) and concentrated by ultrafiltration at 4°C using an Amicon stirred cell equipped with a 10,000 dalton cut-off membrane (YM10) , then 5-20 ml of the concentrate were loaded on a 2.6 cm x 60 cm column of Superdex 200 prep (Pharmacia) attached in series with an identical column (combined dimensions of 2.6 x 120 cm).
  • Both columns were pre-equilibrated with 0.01 M potassium phosphate, pH 7.35, 0.150 M NaCl, 1 x 10" 5 M dithiotreitol at 24°C
  • the chromatography was conducted at a flow rate of 1.5 ml/min; anti-adhesion activity typically eluted 395-410 ml into the run (K 1V of 0.46, see Fig. 2). This elution volume would be expected for a globular protein with a molecular mass of 50,000.
  • the yield of neutrophil function inhibitory activity in this step was typically 70-80%.
  • Concanavalin A-Purified Hookworm Lysate Absorbance at 280 nm is plotted versus elution volumes Active fractions eluted from immobilized Concanavalin A (see step (B) above) and concentrated by ultrafiltration at 4°C using an Amicon stirred cell equipped with a 10,000 dalton cut-off membrane (YM10) , then 5-20 ml of the concentrate were loaded on a 2.6 cm x 60 cm column of Superdex 200 prep (Pharmacia) attached in series with an identical column (combined dimensions of 2.6 x 120 cm).
  • YM10 10,000 dalton cut-off membrane
  • the column was developed with a 50 ml linear gradient of potassium phosphate ranging from 0.001 M to 0.0375 M at a flow rate of 0.5 ml/minute.
  • Neutrophil inhibitory activity eluted sharply at 0.025 M potassium phosphate and then trailed to 0.0325 M potassium phosphate (fractions 37 to 48) .
  • the yield of activity in this step was approximately 48%.
  • Figure 3 depicts Ceramic Hydroxylapatite Chromatography of Superdex/Concanavalin A-Purified Hookworm lysate plotting absorbance at 280 nm and potassium phosphate concentration versus fraction number. Neutrophil inhibitory activity eluted in fractions 37 to 48.
  • Figure 4 depicts the results of reverse phase HPLC of the Neutrophil Inhibitory Factor. Inhibitory activity eluted between 43 and 45% acetonitrile, the activity corresponding with a broad peak at 43-45 minutes.
  • Hookworm lysate was partially fractionated and desalted by molecular sieve chromatography on a 2.6 cm x 60 cm column of Superdex 200 prep (Pharmacia) attached
  • Hookworm lysate fractionated by molecular sieve chromatography on Superdex 75 was mixed with an equal volume of Mono Q buffer [0.02 M Tris-HCl, pH 7.5] and loaded on to a 0.5 x 5.0 cm Mono Q anion exchange column (Pharmacia) equilibrated with Mono Q buffer at a flow rate of 1 ml/minute (306 cm/hour) . The column was then developed with a linear gradient of 0-0.5 M NaCl in column buffer at 0.5 ml/minute (153 cm/hour) . Neutrophil inhibitory activity consistently eluted at 0.4 M NaCl. The overall yield of inhibitory activity for this isolation was about 2-5%.
  • SDS-Polvacrylamide Gel Electrophoresis The protein composition of hookworm lysate and fractionated lysate was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, U.K. 1970, Nature 227, 680) after silver staining (Morrisey, J.H. 1981, Anal. Biochem. 117, 307) . Samples were mixed with an equal volume of 20% glycerol,
  • Lanes 1-10, left to right, are (1) molecular weight standards; (2) molecular weight standards; (3) HPLC pool of HA fractions #37-41, non-reduced; (4) blank; (5) HPLC pool of HA fractions #37-41, reduced; (6) blank, (7) HPLC pool of HA fractions #37-41, reduced, (8) HPLC pool of HA fractions #37-41, non-reduced; (9) HPLC pool of HA trailing fractions #42-48, non-reduced, (l ⁇ )molecular weight standards.
  • the molecular weight standards used were: myosin, 200,000 (rabbit muscle); beta-galactosidase, 116,300 (E.
  • Isolated Neutrophil Inhibitory Factor The estimated mass for the NIF isolated as described in Example 2(E) was determined using laser-desorption time-of-flight mass s ⁇ ectrometry.
  • a 1 ml aliquot of the sample was diluted with an equal volume of a saturated solution of 3,5-dimethozy-4-hydroxy-cinnamic acid dissolved in 30% agueous CH 3 CN, 0.1% TFA.
  • the diluted sample was spotted onto a copper sample stage and allowed to air dry.
  • Mass analysis was performed using a Shimadzu LAMS-50KS laser desorption time of flight mass spectrometer (Shimadzu Corp. , Kyoto, Japan) . Ionization of the sample was accomplished by focusing 500 laser pulses (355 nm, pulse width ⁇ 5 nsec) from a Nd-YAG laser (Spectra-Physics, Inc., Mt. View, CA) onto the sample stage. The resulting ions were accelerated into the mass spectrometer by a 5 kV potential. Calibration of the instrument was accomplished using standard proteins of known mass.
  • Figure 6 depicts the results of laser-desorption time-of-flight mass spectrometry of the isolated neutrophil adhesion inhibitor.
  • Five picomoles of the purified neutrophil function inhibitor was analyzed with a laser desorption time-of-flight mass spectrometer. The estimated mass was determined as 41,200. A small fraction of the sample had a mass of 82,400; this was interpreted to be a dimer.
  • Neutrophil Inhibitory Factor is a Glycoprotein
  • Biotinylated carbohydrate was subsequently detected by reaction with a streptavidin-alkaline phosphatase conjugate. Visualization was achieved using a substrate which reacts with alkaline phosphatase bound to glycoproteins on the membrane, forming a colored precipitate. Neutrophil Inhibitory Factor was stained using this method, demonstrating that it contained carbohydrate and is therefore a glycoprotein.
  • RP HPLC reversed phase HPLC
  • Peptides were isolated by RP HPLC on a ToyoSoda 120T C18 (4.5 X 250 mm) column using an LKB HPLC system with Kratos (ABI, Foster City, CA) detectors.
  • the column was developed with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid (TFA) .
  • TFA trifluoroacetic acid
  • the gradient was from 5 to 54% acetonitrile over 120 minutes at a flow rate of 0.5 ml/minute.
  • Peptide peaks monitored by A 206 and A ⁇ g were collected using an LKB SuperRac with calibrated peak detection.
  • Double stranded cDNA was synthesized from poly(A)+ RNA using random hexa er primers and avian myoblastosis virus (AMV) reverse transcriptase (Amersham, Arlington Hills, IL) .
  • cDNA fragments larger than 1 kilobase pairs were purified on a 6% polyacrylamide gel and ligated to EcoRI linkers (Stratagene) using standard procedures.
  • Linkered cDNA was ligated into lambda gtlO (Stratagene, La Jolla, CA) and packaged using Gigapack Gold II (Stratagene) .
  • Double stranded cDNA probes for hookworm NIF were generated by polymerase chain reaction from hookworm RNA using primers derived from NIF peptide sequences. The sequences obtained for two NIF peptides (see Fig. 7) , T-20 (Leu-Ala-Ile-Leu-Gly-Trp-Ala-Arg) and T-22-10
  • reaction product was amplified using the PCR GeneAmp kit (Perkin Elmer, Norwalk, CT) , with 400 pmol of each of 30.1 and 43.RC (manufactured by Research Genetics, Huntsville, AL) , on a Perkin Elmer DNA Thermal Cycler.
  • PCR conditions were: cycles 1-2, denaturation at 94°C for 2 minutes, annealing at 58°C for 2 minutes and elongation at 72°C for 2 minutes; cycles 3-42, denaturation at 94°C for 45 seconds, annealing at 58°C for 45 seconds and elongation at 72°C for 2 minutes.
  • the -430 base pair amplification product referred to as the 30.2/43.3.RC fragment
  • the 30.2/43.3.RC fragment was separated from reaction contaminants by electroelution from a 6% polyacrylamide gel (Novex, San Diego, CA) .
  • the 30.2/43.3.RC fragment was labelled with [ ⁇ - 32 P]-dCTP (Amersham) using random primer labelling (Stratagene, La Jolla, CA) ; labelled DNA was separated from unincorporated nucleotides using a ChromaSpin-lO column (Clontech, Palo Alto, CA) . Prehybridization and hybridization conditions were
  • NIF phage isolates contained DNA that encoded polypeptides that bore striking resemblance to the amino acid sequences obtained for purified NIF (see Figure 7) .
  • Figure 8 depicts the nucleotide sequence of the coding region of Neutrophil Inhibitory Factor cDNA (clone IFL) and its predicted amino acid sequence.
  • a single isolate, NIF-IFL encoded an open reading frame of 825 nt, initiating with a methionine and terminating with a TGA stop codon ( Figure 8) .
  • NIF polypeptide encoded by NIF-IFL is 274 amino acid residues with a calculated molecular weight of 30,680 daltons.
  • Figure 9 depicts the alignment of the predicted amino acid sequences of several Neutrophil Inhibitory Factor isoform clones. Each line of sequence represents the corresponding sequence segments of the various clones isolated. Each segment is identified by its clone designation (e.g.,
  • IFL, 3P, 2FL, 3FL, 4FL, 6FL and IP The complete amino acid seguence of clone IFL is listed in standard three-letter amino acid code at the top of each sequence segment. Clones having the same amino acid in a given position as clone IFL are denoted by ".”. Amino acid substitutions are indicated by the appropriate three-letter code. " " indicates a space inserted to maintain alignment of the sequences. Tbe carboxy termini of the IFL and IP sequences are denoted by an asterisk.
  • NIF phage isolates encoded partial NIF polypeptides; that is they did not contain either an N-terminal methionine residue or a C-terminal stop codon, as compared to the NIF-IFL polypeptide ( Figure 9) .
  • These partial NIF isolates comprised six predicted NIF isoforms that were significantly similar to, but not identical to the prototypical NIF-IFL polypeptide.
  • the segment of DNA encoding the NIF-IFL isoform was amplified from the original ⁇ gtlO isolate DNA using unique primers for the 5'- and 3'-ends of the coding region.
  • the 5'-primer was composed of a restriction endonuclease site (EcoRl) , a consensus ribosome binding site (Kozak, M. , Cell 44.: 283 (1986)), the ATG initiation codon of NIF and the succeeding 6 codons of the gene.
  • the 3'-primer was composed of a unique nucleotide sequence to the 3'-side of the TGA termination codon of NIF and a restriction endonuclease site (EcoRl) .
  • the nucleotide sequences of the 5'- and 3'-primers were
  • PCR conditions were: cycle 1, denaturation at 97°C for 1 minute, primer annealing for 1 minute at 37°C, ramp from 37°C to 72°C in 2 minutes, and amplification for 2 minutes at 72°C; cycles 3 and 4, denaturation at 94°C for 1 minute, primer annealing for 1 minute at 37°C, ramp from 37°C to 72°C in 2 minutes, and amplification for 2 minutes at 72°C; cycles 5 through 34, denaturation at 94°C for 1 minute, primer annealing for 1 minute at 45°C, and amplification for 2 minutes at 72°C.
  • the amplification product (887 bp) was separated from reaction contaminants using a ChromaSpin 400 column (Clontech Laboratories, Inc. Palo Alto , CA) .
  • the ends of the amplification product were trimmed with the restriction endonuclease EcoRl and the resulting fragment of DNA (875 bp) ligated into EcoRl-digested plasmid pSG5 (Stratagene, La Jolla, CA) using standard techniques.
  • the resulting ligation mixture was used to transform SURE!TM competent cells (Stratagene, .La Jolla, CA) .
  • CRL 1651 by electroporation (0.4 cm electroporation cell, 325 V, 250 F, infinite resistance, 0.5 ml cells at 7 x 10 6 /ml in Hepes buffered saline, pH 7.0, 4°C) . After electroporation the cells were allowed to stand on ice for 2 to 3 minutes before dilution with 14 ml warm DMEM:RPMI 1640 (1 to 1 ratio) supplemented with 10% fetal bovine serum prewarmed to 37°C The cells were placed in 100 mm cell culture dishes and incubated at 37°C with 8% C0 2 . Cell culture supernatant fluid was removed at 1, 2 and 3 days after plating and assayed for NIF activity. (B) Detection and Quantitation of Neutrophil Inhibitory Factor Activity in Cell Culture Medium.
  • Example 15 ml of cell culture fluid was harvested from electroporated C0S7 cells (pSG5/NIFlFLCRl) .
  • this fluid exhibited neutrophil inhibitory activity to dilutions as great as 1:8.
  • An IC j0 at approximately 1:14 was determined using the hydrogen peroxide release assay (Example 1(E)). No activity was observed using cell culture fluid harvested from COS7 cells electroporated with a control expression plasmid (pCAT; Promega, Madison, WI) .
  • (C) Stable Expression in CHO Cells (1) Preparation of Plasmid DNAs
  • the NIF-IFL insert in the pSG5 construct described above in section (A) was excised by digestion with the restriction endonuclease EcoRl.
  • the 875 bp NIF-IFL fragment was gel purified (Magic PC Prep, Promega, Madison, WI) and ligated into EcoRl digested pBluescript II KS (Stratagene, La Jolla, CA) using standard techniques.
  • the resulting ligation mixture was used to transform SURETM competent cells as described by the supplier (Stratagene, La Jolla, CA) .
  • Transformed cells were plated on LB agar containing IPTG and X-gal (Sambrook, Fritsch, and Maniatis, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, pp. 1.85 to 1.86). White colonies were screened for plasmids containing the NIF-IFL insert in the proper orientation by digesting plasmid DNA with BamHI; those colonies harboring a plasmid that yielded a 200 bp BamHI fragment were retained.
  • Plasmid was prepared from one of these colonies (Magic Maxi Prep, ProMega, Madison, WI) and digested with Hindlll and Notl to yield a NIF-IFL fragment with a Hindlll overlap on the 5'-end and a Notl overlap on the 3'-end.
  • This DNA fragment was gel purified and ligated into Hindlll-NotI digested pRC/CMV (Invitrogen, San Diego, CA) .
  • the resulting ligation mixture was used to transform SURETM competent cells.
  • Milligram quantities of pRC/CMV-NIF-lFL were prepared using the Magic Maxi Prep kit.
  • Plasmid DNA was purified from a chloramphenicol amplified culture (Sambrook,
  • the CHO cells were transfected using the Calcium Phosphate Transfection System following the manufacturer's instructions (Gibco BRL, Gaithersburg, MD) in 10 cm cell culture dishes at the following ratios: 1 x 10 6 cells, 20 g pRC/CMV-NIFlFL DNA and 5 g pLTRdHFR26 DNA. The cells were incubated for 12 hr at 37'C in the presence of the co-precipitated DNAs.
  • the dishes were examined for colonies and the culture supernatant fluids assayed for NIF activity using the calcein assay for neutrophil adhesion.
  • the cells in dishes exhibiting NIF activity were trypsinized and washed as before and plated to obtain single colonies.
  • One single cell isolate, designated 8F5, expressing NIF activity was chosen for further methotrexate amplification.
  • 8F5 cells were grown to confluence in a 10 cm cell culture dish, detached with trypsin and washed as before.
  • the cells were diluted with combo medium containing 500 g/ml G418 and 1 x 10° cells were placed in 10 cm culture dishes.
  • the adhered cells were covered with combo medium containing 500 g/ml G418 and 40, 80, 160, 320, or 640 nM methotrexate.
  • the dishes were examined for colonies and supernatant fluids assayed for NIF activity. Cells were released from the plates by trypsin treatment as before.
  • the pool of cells obtained from the 320 nM methotrexate dish was selected for further use and single cell isolates were obtained.
  • the sample was cycled through the column in a closed loop for 1 hour at 2 ml/minute at 20°C.
  • the column was subsequently washed with 5 ml of 0.02 M bis Tris-propane-HCl, pH 7.3, 1 M NaCl, 0.001 M CaCl 2 , 0.001 M MnS0 4 .
  • the buffer resident in the column was displaced with buffer containing 0.5 M methyl-alpha-mannopyranoside and flow stopped- for 15 minutes. Flow was restarted at 1 ml/minute and approximately 11 ml of sugar-containing eluate collected. The eluate was dialyzed 18 hours against 1 liter 10 mM potassium phosphate, pH 7.35, 150 mM NaCl at 4°C and concentrated to 1.1 ml using an Amicon centrifugal concentrator equipped with a 10,000 molecular weight cut-off membrane (CentriPrep 10, Amicon, Beverly, MA) . When assayed by the neutrophil-plastic adhesion assay (Example 1(C)), this sample exhibited substantial activity at a dilution of
  • C0S7(pSG5/NIFlFLCRl) cell culture fluid was dialyzed 18 hours against one liter of 10 mM bis Tris-propane-HCl, pH 7.0 at 4°C and loaded at 3 ml/minute onto a 0.46 x 10 cm column of Poros II Q/M (PerSeptive Biosystems, Inc., League City, TX) equilibrated with the same buffer.
  • the column was washed with one column volume of equilibration buffer and developed with a linear gradient of sodium chloride from 0 to 0.5 M over 14.4 column volumes collecting 2 ml fractions.
  • Pichia shuttle/expression vector A Description of the Pichia shuttle/expression vector.
  • the Pichia strain GTS115 (his4) (Stroman, D.W. et al., U.S. Patent No. 4,655,231 (August ⁇ , 1989)) and the E. coli-Pichia shuttle vectors pHILSl and pHILD5 referred to hereafter are part of the Pichia yeast expression system licensed from the Phillips Petroleum Company (Bartlesville, Oklahoma) .
  • the pHIL7SP8 vector used to direct expression of NIF in P. pastoris was assembled from pHILSl and pHILD5 and from synthetically generated fragments.
  • the pHIL7SP8 plasmid contained the following elements cloned onto pBR322 sequences:
  • This pro-sequence represents one of the two 19-aa pro-sequences designed by Clements et al.,(1991. Gene, 106:267-272) on the basis of the yeast alpha-factor leader sequence.
  • Patent No. 4,655,231 (August ⁇ , 1969) the disclosure of which is incorporated herein by reference) .
  • the segment of DNA encoding NIF was PCR-amplified from a sub-clone of NIF-IFL in Bluescriptll (Stratagene, La Jolla, CA) using unique primers for the 5'- and 3'-ends of the coding region.
  • the 5'-primer contained no restriction endonuclease sites and corresponded to the region beginning at the 5'-end of proteolytically processed NIF and the succeeding 7 codons.
  • the codon for the first residue of the mature NIF was altered from AAT to AAC (both codons translate to asparagine).
  • the 3'-primer was composed of 8 codons at the 3' end of the coding region, a TAA stop replacing the TGA stop of the natural gene, and three unique restriction endonuclease sites (Hindlll. Spel, and Bglll) .
  • the sequences of the 5'- and 3'-primers used were 5'-AAC-GAA-CAC-AAC-CTG-AGG-TGC-CCG and
  • Amplification was accomplished using 100 pmol of each primer, 2 units of Vent polymerase in IX Vent buffer (New England Biolabs, Beverly, MA), and 0.2 mM of each of dATP, dCTP, dGTP, and dTTP.
  • One hundred nanograms of Bluescriptll-containing NIF-IFL were used as template DNA.
  • the PCR conditions were the same for all ten cycles: denaturation at 95°C for 1 minute, primer annealing at 60°C for 1 minute, and amplification for 1.5 minutes at 72°C.
  • the amplification product was purified as described above and digested with Bglll. The amplification product was then ligated into Stul-BgJ.il cleaved pHIL7SP8 using standard methods.
  • the ligation mixture was used to transform E.coli WK6, and ampicillin resistant clones were obtained on ampicillin plates. Based on restriction and DNA sequence analysis, correct insert sequences in two of the resulting plasmid clones, pHIL7SP-NIlcl and pHIL7SP-NIlcl0, were selected to transform the P.pastoris yeast strain GTS115 (his4) . These vectors were digested with either Notl (targeting integration to the expression cassette in the AOXl region) or Sail (targeting integration to the HIS4 locus) . The 4 restricted DNA preparations were introduced individually into Pichia by electroporation, essentially as described by Becker, D. and Guarente, L. , Methods in Enzymology, vol.
  • the cells were grown in YEPD medium at 30°C to an 00 ⁇ of 1.3 to 1.5.
  • the cells were pelleted at 4°C (1500 x g for 5 minutes) and resuspended in 500 ml ice cold sterile distilled water.
  • the cells were pelleted as above and resuspended in 250 ml ice cold distilled water.
  • After a final pelleting the cells were resuspended in 1 ml ice cold 1 M sorbitol.
  • Forty ⁇ l cells in 1 M sorbitol were mixed with 5 ⁇ l of linearized DNA and the mixture transferred to an ice cold 0.2 cm gap electroporation cuvette. After 5 minutes on ice, the cells were pulsed at 50 uF,
  • Pichia cell supernatant (pHIL7SP-NlclO) was obtained by centrifugation for 15 minutes at 1,800 x g max from cells 48 hours following methanol induction and filtered through a 0.22 ⁇ m cellulose acetate membrane.
  • the filtered cell supernatant solution was concentrated about 3-fold using centrifugal concentrators equipped with a 10,000 MWCO membrane (Amicon MicroCon 10, Beverly, MA) and desalted by gel filtration using a 1 x 10 cm column of G-25 Sephadex Superfine (Pharmacia,
  • Pichia cell supernatant (pHIL7SP-NlclO) 48 hours following methanol induction was obtained by centrifugation for 15 minutes at 1,800 x g,, ⁇ and filtered through a 0.22 ⁇ m cellulose acetate membrane.
  • This concentrate was dialyzed at 4°C for 6 hours against one liter of 0.05 M bis Tris-propane-HCl, pH 7.0 to adjust the pH to neutrality, and then against two changes of one liter of 0.001 M potassium phosphate, pH 7.0.
  • Fractions exhibiting substantial neutrophil inhibitory activity were combined and concentrated to about 3 ml using an Amicon centrifugal concentrator equipped with a 10,000 molecular weight cut-off membrane (CentriPrep 10, Amicon, Beverly, MA) and applied to a 1 x 25 cm C4 300 A reverse phase column (5 ⁇ m particle size, Vydac, Hesperia, CA) equilibrated with 0.1% trifluoroacetic acid. The column was washed with four column volumes of equilibration buffer and then developed with a linear gradient of acetonitrile from 15 to 40% ⁇ ver 10 column volumes at a flow rate of 5 ml/min.
  • the activity of the inhibitor was assessed in a non-neutrophil cell adhesion-based assay, platelet aggregation.
  • the effects of the hookworm Neutrophil Inhibitory Factor on blood platelet aggregation were examined. Platelet aggregation was performed with human platelet-rich plasma (PRP) . PRP was stirred at 37°C in an aggregometer (Scienco Model 247, Morrison, CO) and aggregation was initiated by the addition of _b0 ⁇ M ADP (Sigma, St. Louis, MO) .
  • a concentration of Neutrophil Inhibitory Factor of approximately 150 nM a concentration that completely blocked neutrophil function as assessed by neutrophil-HUVEC and neutrophil-plastic adhesion assays, homotypic neutrophil aggregation and hydrogen peroxide release by neutrophils, had no inhibitory effect on ADP-induced aggregation of human platelets.
  • CDllb/CDl8 Integrin is a Primary Receptor for Neutrophil
  • NIF purified from Ancylostoma canirium was radiolabeled using the following method. Approximately 30 ⁇ g NIF was labeled with 2 mCi Na 125 I (carrier free; Amersham, Arlington Hills, IL) using Enzymobeads (BioRad, Hercules, CA) Briefly, to a 1.5 ml eppendorf test tube was added 360 ⁇ l of the Enzymobead suspension together with 180 ⁇ l of a 1% beta-D-glucose solution, NIF and Na 125 I. This mixture was allowed to react at room temperature for 30 minutes.
  • NIF labeled NIF was separated from unbound 125 I-iodine by desalting on a PD10-DG column (BioRad, Hercules, CA) using phosphate buffered saline (0.1 M sodium phosphate pH 7.2, 0.15 M sodium chloride) containing 1% bovine serum albumin as elution buffer. Radioactive fractions containing NIF were pooled. The specific activity of the 125 I-NIF was 13.9 ⁇ Ci/ ⁇ g.
  • Various leukocyte proteins were assessed for ability to capture NIF in immunoprecipitation experiments. Potential cellular receptors for NIF were selected from a detergent extract of leukocytes using specific monoclonal antibodies.
  • Leukocytes were prepared from human blood using Mono-poly (ICN, Biomedicals Inc., Costa Mesa, CA) .
  • the leukocyte cell pellet was resuspended in 1 ml resuspension buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl 2 ) followed by the addition of 1 ml extraction buffer (2% Triton X-100, 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl 2 ) .
  • 1 ml extraction buffer 2% Triton X-100, 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM CaCl 2
  • Monoclonal antibody-test protein complexes were formed by incubating 10 ⁇ g specific monoclonal antibody with 200 ⁇ l of leukocyte detergent extract at 4°C for 4 hours. To this mixture was added 2.5 ⁇ l of the 125 I-NIF and these reagents were incubated at 4°C for 18 hours.
  • Precipitation of the complex was effected by adding this mixture to a 1.5 ml eppendorf test tube containing 50 ⁇ l of protein G-sepharose (Pharmacia, Pistdcaway NJ; resuspended in TACTS 20 buffer (0.05% Tween 20, 20 mM Tris pH 8, 120 mM NaCl, 2 mM CaCl 2 ) with 1% bovine serum albumin) and gently agitating at 4°C for 2 hours. The protein G-sepharose beads were subsequently washed four times with TACTS 20 buffer.
  • protein G-sepharose Pharmacia, Pistdcaway NJ; resuspended in TACTS 20 buffer (0.05% Tween 20, 20 mM Tris pH 8, 120 mM NaCl, 2 mM CaCl 2 ) with 1% bovine serum albumin
  • 125 I-NIF did not precipitate when MAbs to other leukocyte integrins were used including MAbs directed against the VLA-4 (L25.3; Becton Dickinson, Sunnyvale, CA) and CDllc/CDl ⁇ (SHCL-3; Becton Dickinson, Sunnyvale, CA) integrin complexes.
  • a relatively minor amount of 125 I-NIF was observed when a MAb directed against the CDlla/CD18 (TS1/22; ATCC# HB202) integrin complex was used. This was likely due to cross-reactivity of the anti-CDlla/CD18 antibody with the related integrin complex CDllb/CD18.
  • CDllb/CD18 is a cell-surface receptor f r Ancylostoma caninum NIF on leukocytes.
  • biotin-labeled NIF was used to precipitate NIF-associating proteins from a detergent extract of surface iodinated leukocytes.
  • NIF was biotinylated by conjugation to its carbohydrate moieties. Approximately 16 ⁇ g of NIF purified from hookworm (Ancylostoma caninum) lysates (hydroxyapatite eluate; see Example 2(D)) was oxidized with 50 mM NaI0 4 in 1 ml 0.1 M sodium acetate, pH 5.5. After 20 minutes at 4°C the reaction was terminated with the addition of 100 ⁇ l 165 mM glycerol. Oxidized NIF was separated from other reaction products using a Microcon 10 concentrator (Amicon, Beverly, M* , and diluted into 100 ⁇ l 0.1 M sodium acetate, pH 5.5.
  • Biotinylation was effected by the addition of 400 ⁇ l 6.25 mM biotin-LC-hydrazide (Pierce, Skokie, IL) . The reaction was allowed to proceed for 18 hours at 4°C Biotinylated NIF was worked up by buffer exchange into phosphate buffered saline (PBS; 0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2), using a Microcon 10 concentrator. To 250 ⁇ l of the concentrate was added an equal volume of glycerol, giving a final NIF-biotin concentration of approximately 32 ⁇ g/ml. This material was stored at -20°C
  • anti-CD18 integrin complex monoclonal antibodies LM-2 and OKM-l anti-CDllb/CD18; ATCC #HB204 and CRL8026, respectively
  • TS1/22 anti-CDlla/CD18; ATCC# HB202
  • a -total leukocyte fraction prepared from 90 ml of fresh human blood using Mono-Poly density gradient separation (ICN Biomedical, Costa Mesa, CA) , was suspended in 0.5 ml phosphate buffered saline. To the cell suspension was added 2 mCi Na 125 I (carrier free; Amersham; Arlington Heights, IL) , 60 ⁇ l 0.03% hydrogen peroxide and 100 ⁇ l lactoperoxidase at 2 mg/ml (BioRad; Hercules, CA) . The reaction was allowed to proceed for 30 minutes at room temperature, with gentle agitation every two minutes.
  • the reaction was terminated by the addition of 25 mM KI in PBS, and the cells were washed two times with PBS.
  • the leukocyte cell pellet was resuspended in 1 ml resuspension buffer and leukocyte extract was prepared as described above in Example 14(A) .
  • NIF-biotin 32 ⁇ g/ml was diluted with 40 ⁇ l resuspension buffer and incubated with 200 ⁇ l 125 I-labeled leukocyte extract at room temperature for 6 hours.
  • NIF-associating proteins from the leukocyte extract was effected by the addition of 100 ⁇ l streptavidin-agarose (Pharmacia; Piscataway, NJ) to this mixture.
  • Test tubes were agitated gently for 18 hours at 4°C Beads were subseguently washed four times with 500 ⁇ l TACTS-20 buffer (0.05% Tween 20, 20 mM Tris pH 8, 120 mM NaCl, 2 mM CaCl 2 ) , and associated proteins were solubilized with 50 ⁇ l sample buffer (5% /3-mercaptoethanol) and analyzed by SDS-PAGE as described in Example 5.
  • Control precipitations were performed in a similar manner with biotinylated monoclonal antibodies to CDllb/CD18 and CDlla/CDl ⁇ .
  • Biotinylated NIF precipitated two 125 I-labeled polypeptides that, when separated by 6% SDS-PAGE, had apparent molecular weights of about 170 kDa and about 95 kDa. These polypeptides comigrated on SDS-PAGE in this experiment with the two polypeptides that were precipitated by the anti-CDllb/CD18 monoclonal antibodies LM-2 and OKM-l. This data strongly suggests that CDllb/CD18 is a major receptor for NIF on leukocytes when considered with the results of the previous experiment (Example 14(A)), in which CDllb/CDl8 was shown to associate with NIF.
  • Toxocara canis Frozen canine worms Toxocara canis were obtained from Antibody Systems (Bedford, TX) and were stored at -70°C until homogenized. Toxocara canis were homogenized on ice in homogenization buffer [0.02 M Tris-HCl pH 7.4, 0.05 M NaCl, 0.001 M MgCl 2 , 0.001 M CaCl 2 , 1.0 X 1(T 5 M E-64 Protease Inhibitor (CAS 66701-25-5), 1.0 X 10 "6 M pepstatin A (isovaleryl-Val-Val-4-amino-3-hydroxy-6-methyJ--heptanoyl -Ala-4-amino-3-hydroxy-6-methylheptanoic acid, CAS 26305-03-3), 1.0 X 10" 5 M chymostatin (CAS 9076-44-2), 2.0 X 10" 5 M APMSF (amidinophenylmethylsulfony
  • protease inhibitors E64, pepstatin A, chymostatin, and APMSF were obtained from Calbiochem (La Jolla, CA) .
  • Approximately 3-6 ml of homogenization buffer was used to homogenize each gram of frozen worm. Twenty-four grams of worms was used in total.
  • Insoluble material was pelleted by two sequential centrifugation steps: 40,000 X g-- ⁇ at 4°C for 25 minutes followed by 105,000 X g-, ⁇ at 4°C for 1 hour.
  • the supernatant solution was clarified by passage through glass wool and a 0.45 ⁇ m cellulose acetate filter (CoStar, Cambridge, MA) .
  • Toxocara canis lysate (66 ml) was absorbed to 26 ml of Concanavalin A Sepharose (Pharmacia, Piscataway, NJ) pre-eguilibrated with Con A buffer [0.02 M Tris-HCl, pH 7.4, 1 M NaCl, 0.001 M CaCl 2 , 0.001 M MnS0 4 ] by recycling it through a 1.6 X 13 cm column at a flow rate of 4 ml/minute (119 cm/hour) for 2 hours.
  • the column was at room temperature (24°C) while the reservoir of lysate was maintained on ice throughout the procedure.
  • the column was subsequently washed with 100 ml of Con A buffer. Material that had activity in anti-adhesion assays (see, Section (D) below) was eluted with approximately 3-5 column volumes of Con A buffer containing 0.5 M methyl-alpha-mannopyranoside (CAS)
  • Neutrophil Inhibitory Factor isolated from canine hookworms was tested in an animal model of acute inflammation.
  • Peritoneal inflammation was induced in 150-250 gram Sprague-Dawley rats by an intraperitoneal injection of nine ml of 2% oyster glycogen in H 2 0 (see Baron et al., Journal of Immunological Methods. 4jJ:305, 1982; McCarron et al., Methods in Enzvmology. 108:274, 1984; Feldman et al., Journal of Immunology. 113:329, 1974; Rodrick et al., Inflammation. .6:1, 1982; and Kikkawa et al., Laboratory Investigation. 3Q:76, 1974).
  • NIF was prepared as described in Example 2. Lysate from approximately 20,000 hookworms (48.2 g wet weight) was prepared and chromatographed on ConA, Superdex, and hydroxyapatite (HA) . The active fractions from two equivalent HA runs were combined to yield 41 ml of HA material. One ml of NIF solution (11 ⁇ g) was administered simultaneously with the glycogen by the intraperitoneal route or thirty minutes prior to glycogen administration by the intravenous route.
  • FIG. 10 depicts the effects of varying doses of Neutrophil Inhibitory Factor isolated from canine hookworms on neutrophil infiltration in peritoneal inflammation in rats induced by interperitoneal infusion with glycogen.
  • NIF glycogen-induced rat peritoneal inflammation
  • NIF and glycogen were administered by the intraperitoneal route as previously described.
  • 1 ⁇ g of NIF was administered intravenously thirty minutes prior to the intraperitoneal infusion of glycogen.
  • a third group of animals received glycogen and NIF treatment was replaced with saline. Four hours later the peritoneal exudate was collected and blood cells were counted.
  • Figure 11 depicts the effect of Neutrophil
  • Inhibitory Factor isolated from canine hookworms on neutrophil infiltration in peritoneal inflammation in rats induced by intraperitoneal infusion of glycogen.
  • Neutrophil Inhibitory Factor (1 ml) was injected by intraperitoneal route in conjunction with intraperitoneal infusion of glycogen, or by intravenous route thirty minutes prior to infusion of glycogen.
  • Figure 11 represents a summary of the six independent experiments for the intraperitoneal administration of NIF and the results of the single experiment for the intravenous administration of NIF. These results demonstrate that NIF, when administered by either the intraperitoneal or intravenous route, was effective in the prevention of peritoneal inflammatory response in glycogen-stimulated rats.
  • rNIF recombinant NIF
  • inflammation was induced in the rat ear by topical administration of arachidonic acid.
  • Sprague-Dawley rats 250g were anesthetized with pentobarbital (initial dose of 65 mg/kg intraperitoneal; Anpro Pharmaceutical, Arcadia, CA) ; rats were maintained at a surgical plane of anesthesia for the duration of the experiment (4 hours) .
  • a catheter was inserted into the femoral vein of the anesthetized rat.
  • One hundred microliters of recombinant NIF produced in Pichia pastoris; see Example 12
  • Control rats received 100 ⁇ L sterile 0.14 M NaCl.
  • arachidonic acid (Sigma, St. Louis, MS; diluted 1:1 with acetone to a final concentration of 500 mg/ml) was applied to the right ear in three 10 ⁇ l applications each to the ijnside and the outside of the ear. The right ear thus ⁇ received a total dose of 30 mg arachidonic acid.
  • the left ear used as a background control, received a total of 60 ⁇ l acetone.
  • the rat was sacrificed with C0 2 .
  • Neutrophil infiltration into the arachidonic acid-treated ear tissue was quantitated indirectly by determining myeloperoxidase activity.
  • a tissue sample was obtained from the center of each ear using a 7 mm skin punch (Miltex; Lake Success, NY) .
  • the tissue sample was cut into small pieces and added to a 16 x 100 mm test tube that contained 0.5 ml HTAB buffer (0.5% hexadecyltrimethylammonium bromide in 50 mM sodium phosphate, pH 6.4; HTAB was purchased from Sigma, St. Louis, MO) .
  • the ear tissue was homogenized for 20 seconds using an Ultra-Turrax (Janke and Kunkel; Staufen, Germany) at high speed.
  • Insoluble matter was removed from the homogenate by centrifugation. at 14,000 x g for 10 minutes followed by filtration through Nytex gauze. Myeloperoxidase determinations were done in triplicate in 96 well polystyrene plates (Costar; Cambridge, MA) . Twenty five microliters of HTAB-solubilized ear tissue was added to each well, and to this was added 100 ⁇ l of substrate solution.
  • Substrate solution comprised two components: (1) 0.012% H 2 0 2 in 0.1 M sodium acetate pH 4.5 and (2) 0.3 mg/ml 3,3' ,5,5'-tetramethylbenzidine in 10% HCl, combined immediately prior to use at a ratio of 0.125:1.
  • Recombinant NIF had a protective effect on arachidonic acid-induced neutrophil infiltration into ear tissue.
  • One myeloperoxidase unit will produce an increase in absorbance at 470 nm of 1.0 per minute at pH 7.0 and 25°C, calculated from the initial rate of reaction using guaiacol as substrate (Desser, R.K.
  • Genomic DNA or cDNA libraries are formed using standard procedure (for example see Molecular Cloning. A Laboratory Manual. Sambrook, J., Fritsch, EF., and
  • libraries may be from any animal, fungal, bacterial or viral source, such as Ancylostoma caninum. other Ancylostoma species, other helminths and mammals including human placental tissue.
  • Such libraries are screened for useful clones by nucleic acid hybridization using NIF cDNA sequences isolated from Ancylostoma as probe.
  • NIF cDNA fragments of about 100-2000 base pairs labeled for detection by standard procedure (for example, see Molecular Cloning. A Laboratory Manual. Sambrook, J., Fritsch, EF., and Maniatis, T. 2nd Ed. Cold Spring Harbor Laboratory Press, CSH, NY 1989) is hybridized with a library from another tissue or another species under conditions of variable stringency.
  • reduced stringency hybridization conditions are utilized (eg 6X SSC [SSC is 150 mM NaCl, 15 mM trisodium citrate], 0.02 M sodium phosphate pH 6.5, 5X Denhardt's solution, 0.5% (w/v) SDS, 0.01 M EDTA, 100 ⁇ g/ml sheared, denatured salmon sperm DNA, 0.23% dextran sulfate, 20-30% formamide at 42°C for 18 hours) .
  • reduced stringency conditions are used to wash filters after hybridization (0.5 to 2X SSC at 45-60°C for 20 minutes after two prewashes with 2X SSC for 15 minutes) .
  • NIF-related complementary DNAs isolated using the techniques described above are subjected to nucleotide sequence analysis using the procedure of dideoxy sequencing (Sanger et al, 1977, Proc. Natl. Acad. Sci. USA 24:5463-5467).
  • Isolates containing open reading frames i.e., initiating with a methionine and terminating with a TAA, TGA or TAG stop codon
  • suitable vectors for protein expression in either bacterial, yeast, insect or mammalian cells are inserted into suitable vectors for protein expression in either bacterial, yeast, insect or mammalian cells.
  • Expression systems comprise vectors designed to secrete recombinant protein (i.e., fusion of cDNA isolate open reading frame with a known secretion signal sequence for that cell type) into the culture medium.
  • Vectors lacking a homologous secretion signal sequence are also used for expression. Either conditioned media or cell lysate, depending on the expression system used, is tested for inhibitory activity using one or more of the following criteria for neutrophil activaftion: release of hydrogen peroxide, release of superoxide anion, release of myeloperoxidase, release of elastase, homotypic neutrophil aggregation, adhesion to plastic surfaces, adhesion to vascular endothelial cells, chemotaxis, transmigration across a monolayer of endothelial cells and phagocytosis.
  • Proteins that are structurally related to NIF and that are inhibitory in one or more of these neutrophil function assays would be considered to belong to the NIF family of related molecules.
  • primers are engineered so that this fragment contains 5' and 3' restriction sites that are compatible with insertion into the selected expression vector.
  • the expression construct is preferably engineered so that the recombinant NIF will be secreted into the cytoplasm and not the periplasmic space. This may be accomplished by omitting an ______ coli secretion signal from the construct.
  • ______ coli cells are transformed with «*the NIF-IFL expression vector construct using standard methods.
  • Cells are grown in appropriate media (e.g. Luria Broth; see Molecular Cloning. A Laboratory Manual, Sambrook, J. Fritsch, E.F. and Maniatis, T., Second Edition, Cold Spring Harbor Laboratory Press, 1989, A.l) and harvested before they reach the stationary phase of growth.
  • the majority of the recombinant NIF should be present in the cytoplasm in the form of insoluble and functionally inactive aggregates.
  • the solubilization and refolding of the recombinant protein present in these aggregates may be accomplished using known methods such as those reviewed in detail in Kohno et al., 1990 (Methods in Enzymology, 185:187-195).
  • Refolded recombinant NIF may be separated from unfolded recombinant NIF and other reaction products using a number of standard chromatographic techniques, including C4 reverse phase HPLC (see, e.g.. Example 2(E) ) .
  • Refolded recombinant NIF is tested for functional activity using the neutrophil function assays described in Example 1.
  • This recombinant NIF is not glycosylated.
  • PCR oligonucleotide primers were designed to generate an amplification product that contains the
  • the PCR product initiates at the first Asn-codon of mature NIF-IFL which ⁇ as a result of the amplification was changed from AAT to AAC
  • the 3'-primer replaces the TGA translational stop codon by a TAA triplet and introduces a Spel, a Hindlll and a Bglll site downstream of the coding region.
  • the PCR primers were as follows:
  • the expression module consists of the following elements: (1) The phage lambda P R promoter.
  • the leader-cistron has the potential to code for a 31 residue polypeptide.
  • a SD-sequence (eg, GGAGGT; see Figures 14 and 15) is present.
  • fdT phage fd derived transcription terminator
  • the vector was introduced in W3110 cells harboring pcI857.
  • the plasmid pcI857 specifies resistance to kanamycin (20 ⁇ g/ml) ,. encodes a temperature sensitive repressor of the lambda P R promoter, and is compatible with the pMa5-NIl/3.
  • Cultures were grown in LB medium at 2 ⁇ "C to a density of about 2 x 10 8 cells/ml and then induced at 42 * C for 2-3 hours.
  • the cell pellets were frozen at -80 ⁇ C Each tube contained about 3.5 g cells. Fifteen milliliters of TES buffer (0.05 M Tris, 0.05 M sodium ethylenediaminetetraacetate, 15% (w/v) sucrose, pH 8.0) was added to one tube, and the tube was sonicated to thaw and disperse the cell pellet. The suspension was then distributed into two 30 ml glass centrifuge tubes. An additional 2.5 ml of TES was used to wash the original tube and this wash solution was added to the glass tube.
  • TES buffer 0.05 M Tris, 0.05 M sodium ethylenediaminetetraacetate, 15% (w/v) sucrose, pH 8.0
  • the suspensions in the glass tubes were sonicated (Branson Sonic Power Co., Danbury, Connecticut) four times for 30 seconds each, with an ice incubation between sonications to maintain the temperature ⁇ 10°C throughout the procedure.
  • the tubes were then centrifuged at 10,000 rpm for 20 minutes (12,100 x g,, ⁇ ) at 4°C The supernatants were discarded.
  • the pellets were resuspended in 15 ml of PSX buffer
  • the pellets were resuspended in 15 ml of PBS buffer (0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.3) per tube, briefly sonicated, and centrifuged as before. The PBS resuspension was repeated one additional time. The pellets were then resuspended in PBS by sonication, 12.5 ml per tube. The contents of both tubes were combined, and the volume was brought to 30 ml by the addition of further PBS. The protein concentration of the purified inclusion bodies was determined using the DC Protein Assay (Bio-Rad, Hercules, California) .
  • the tubes were vortexed at 37 * C for 48 hours using a Thermomixer vortexer (Eppendorf, Hamburg, Germany) . Following this incubation, samples were submitted for NIF activity assays (see Example 1) . Typically, activity corresponding to the activation (refolding) of 3% of the NIF present was found.
  • YG2 5'-TCA-TAA-CTC-TCG-GAA-TCG-ATA-AAA-CTC (3'-primer matching with C-terminal sequence corresponding to: E-F-Y-R-F-R-E-L-Stop codon)
  • the YG1/YG2 primer couple was found to yield a correctly sized PCR product when using a total RNA preparation of Aj_ caninum (see Example 10) as template.
  • First strand cDNA synthesis (First-Strand cDNA Synthesis Kit of Pharmacia, Uppsala, Sweden; 10 pmoles of the YG2 primer; -15 ⁇ g of total RNA) and the subsequent amplification by PCR were carried out according to the manufacturer's specifications.
  • PCR-NIF5 was found to be identical to NIF-IFL.
  • PCR-NIF7 and PCR ⁇ IF20 represent two new NIF sequences.
  • Poly(A+) RNA was prepared from adult worms using the QuickPrep mRNA Purification Kit (Pharmacia, Uppsala, Sweden) . Using this poly(A+) RNA preparation as template, an amplification product of about the expected length was obtained with the YG3/YG4 primer couple (the PCR conditions were as descibed above) . The amplification product was shown by gel-electrophoretic analysis to be rather heterogeneous with respect to length. The YG3/YG4 primers were indeed designed to target sequences that flank that part of the coding region where the various NIF sequences display significant differences in length; the heterogeneous nature of the PCR product indicated that the primers are useful for the amplification of several different isoforms.
  • PCR product were gel-purified and subseguently radiolabeled by "random primer extension” ( " “QuickPrime KitTM; Pharmacia, Uppsala, Sweden) for use as hybridization probe.
  • a cDNA library was constructed using described procedures (Promega Protocols and Applications Guide 2nd Ed.; Promega Corp.). About 3 ⁇ g of mRNA was reverse transcribed using an oligo(dT)-Notl primer-adaptor [5'-TCGCGGCCGC(T) 1S ; Promega Corp., Madison, WI] and AMV (Avian Myeloblastosis Virus) reverse transcriptase (Boehringer, Mannheim, Germany) .
  • the enzymes used for double stranded cDNA synthesis were the following: E. coli DNA polymerase I and RNaseH from BRL Life Technologies (Gaithersburg, MD) and T4 DNA polymerase from Pharmacia.
  • the obtained cDNA was treated with EcoRl methylase (RiboClone EcoRl Linker Ligation System; Promega) .
  • the cDNAs were digested with Notl and EcoRl, size selected on a 1% agarose gel (fragments of between 1000-7000 base-pairs were eluted using the Geneclean protocol, BIO101 Inc., La.
  • cDNA library The usefulness of the cDNA library was demonstrated by PCR analysis (Taq polymerase from Boehringer; 30 temperature cycles: 1 minute 95°C; l minute 50"C; 3 minutes 72 * C) of a number of randomly picked clones using the lambda gtll primer #1218 (New England Biolabs, Beverly, MA) in combination with the above mentioned oligo(dT)-Notl primer adaptor. The majority of the clones was found to contain cDNA inserts of variable size.
  • the segments of DNA encoding AcaNIF24, AcaNIF6, AcaNIF4, and AcaNIF9 were PCR amplified using pGEM-type vectors containing the respective cDNAs (see above) as template.
  • the 5'-primers contained no restriction sites and matched with the 5'-end of that part of the coding regions corresponding to the mature protein. Also, the first codon was altered from AAT to AAC (both codons translate to asparagine) .
  • the sequences of the 5'-primers for the various NIF sequences were as follows:
  • AcaNIF24 5'-AAC-GAA-CAC-AAC-CTG-ACG-TGC-CC
  • AcaNIF ⁇ 5'-AAC-GAA-CAC-AAA-CCG-ATG-TGC-CAG-C
  • AcaNIF4 5'-AAC-GAA-CAC-AAA-CCG-ATG-TGC-GAG
  • the 3'-primers were composed of ⁇ codons at the 3'end of the coding region, a TAA stop replacing the TGA stop of the natural gene, and three unique restriction endonuclease sites (Spel, Hindlll, and Bglll) .
  • the 3'-primers used were:
  • Amplification was accomplished using 100 pmoles of each primer, 2 units of Vent polymerase in lx Vent buffer (New England Biolabs, Beverly, MA), 0.2 mM of each dNTP and 100 ng of template DNA.
  • the PCR conditions were the same for all twenty cycles: denaturation at 95'C for 1 minute, primer annealing at 60"C for 1 minute, and amplification for 1.5 minutes at 72°C
  • the amplification product was gel-purified and digested with Spel.
  • the amplification product was ligated into Stul-Spel cleaved pHIL7SP8 using standard methods. The ligation mixture was used to transform E. coli WK6 selecting for ampicillin resistant clones.
  • Recombinant AcaNIF4 was purified by hydroxyapatite and reverse-phase chromatography essentially as described in Example 23, however, the gel filtration step on Superdex was omitted. The purified protein was found to migrate as a single band on SDS-PAGE (4-20% gradient gel; Novex) . The concentration was determined spectrophotometrically. The results obtained in a competitive binding assay with biotinylated NIF-IFL indicated that AcaNIF4 had a significantly lower affinity for the LM2/CDllb/CD18 complex than NIF-IFL ( Figure 18) .
  • Recombinant AcaNIF9 was partially purified by reverse-phase chromatography (see Example 23) - Edman degradation revealed N-E-H-D-P as N-terminal amino acid sequence confirming that correctly processed AcaNIF9 protein was produced.
  • This partially purified protein was found to have a considerably higher mobility on SDS-PAGE (4-20% gradient gel; Novex) than the
  • NIF-IFL protein (30-35 kDa compared to 40-80 kDa) consistent with the presence of seven N-glycosylation sites in NIF-IFL and of only two potential N-glycosylation sites in AcaNIF9.
  • the sample containing AcaNIF9 was tested in the competitive binding assay described in Example 1. The results demonstrated that binding of biotinylated recombinant NIF-IFL to the LM2/CDllb/CD18 complex can be prevented by recombinant AcaNIF9.
  • A Cloning and Seguencing of NIF Sequences From A. ceylanicum.
  • a full-length A. ceylanicum NIF gene was isolated by screening a cDNA library using as hybridization probe a PCR fragment effected from the same species. The PCR fragment was obtained using primers that target sequences which are highly conserved among the seven A. caninum NIF isoforms described in Example 10 (IFL, 3P, 2FL, 3FL, 4FL, 6FL and IP) . These primers, designated YG3 and YG4, are described in Example 20.
  • Poly(A-t-) RNA was prepared from A. ceylanicum adult worms using the QuickPrep mRNA Purification Kit
  • the PCR was carried out with Taq DNA polymerase (Boehringer, Mannheim, Germany) , 100 pmoles of both YG3 and YG4 and using 30 temperature cycles (1 minute denaturation step at 95°C; 1 minute annealing period at 55°C; 1.5 minutes elongation step) .
  • the PCR product was gel-purified and subsequently radiolabeled by "random primer extension" ("QuickPrime KitTM; Pharmacia) for use as hybridization probe.
  • An A. ceylanicum cDNA library was constructed in lambda gtll using the procedures described in Example 20.
  • the quality of the cDNA library was demonstrated by PCR analysis (Taq polymerase from Boehringer; 30 temperature cycles: 1 minute at 95 * C; 14 ⁇ inute at 50"C; 3 minutes at 72°C) of a number of randomly picked clones using the lambda gtll primer #1218 (New England Biolabs) in combination with an oligo(dT)-Notl primer adaptor (Promega) .
  • the majority of the clones were found to contain cDNA inserts of variable size.
  • the A. ceylanicum AceNIF3 region coding for the mature protein was cloned onto a phage display vector according to the procedures described for NIF-IFL in Example 22.
  • the N-terminal amino acid sequence of the authentic A. ceylanicum NIF protein is not known; it is, therefore, difficult to unambiguously locate the secretion signal processing site on the deduced amino acid sequence ( Figure 19) .
  • the following oligonucleotide primers were chosen to PCR amplify the
  • AceNIF3 coding region
  • A-GCAG (54-mer; 5'-primer; the Ncol site and AceNIF3 N-terminus are underlined);
  • the resultant vector designated pAN-AceNIF3 contains the intended in-frame fusion of the pelB, AceNIF3, and M13 gill coding regions.
  • Phages displaying the AceNIF3 protein were obtained by infecting TGl(su + ) bacteria harboring pAN-AceNIF3 with M13-VCS 'helper'-phage (see procedures described in Example 22) .
  • the rescued phages, resuspended in PBS, were assayed by an ELISA on immobilized LM2/CDllb/CD18 complex (see Figure 20) .
  • Phages displaying the A ⁇ . caninum NIF-IFL isoform (Example 22) were used as positive control.
  • NIF-IFL- and AceNIF3-displaying phages Displacement of both NIF-IFL- and AceNIF3-displaying phages by an increasing amount of soluble Pichia produced recombinant NIF-IFL demonstrates that both NIF proteins bind to the same site on the CDllb/CD18 receptor with a comparable affinity. Phage display of the AceNIF3 protein was also demonstrated by Western blot. After fractionation on an SDS-10% polyacrylamide gel, phage proteins were transferred onto ProBlott membrane (Applied Biosystems Inc.) and incubated consecutively with a rabbit anti-pglll serum (GATC GmbH, Konstanz, Germany) and goat anti-rabbit alkaline phosphatase conjugate. Bands corresponding to the wild type phage pglll protein and to the NIF-pglll fusion product could be visualized.
  • the segment of DNA encoding AceNIF3 was PCR amplified from a subclone of AceNIF3 in pGEM-9Zf(-) (see above) using unique primers for the 5'- and 3'-ends of the coding region.
  • the 5'-end of the proteolytically processed AceNIF3 being not unambiguously defined, a hybrid 5'-end was created based on sequence homology between AcaNIF9 and AceNIF3: the three N-terminal codons of proteolytically maturated AcaNIF9 were used as 5'-end, followed by six codons originating from the AceNIF3 sequence.
  • the resulting N-terminal amino acid hybrid sequence was: N-E-H-E-P-T-C-K-Q, while the natural AcaNIF9 sequence was N-E-H-D-P-T-C-P-Q, and the natural AceNIF3 sequence was K-G-D-E-P-T-C-K-Q.
  • the sequence of the 5'-primer used was 5'-AAC-GAA-CAC-GAA- CCA-ACG-TGC-AAG CAG.
  • the 3'-primer was composed of 8 codons at the 3'-end of the coding region, a TAA stop replacing the TGA stop of the natural gene, and three unique restriction endonuclease sites (Spel.
  • Amplification was accomplished using 100 pmoles of each primer, 2 units of Vent polymerase in lx Vent buffer (New England Biolabs, Beverly, MA), and 0.2 mM of each of dATP, dCTP, dGTP, and dTTP.
  • One hundred nanograms of pGEM-9Zf(-) containing AceNIF3 were used as template DNA.
  • the PCR conditions were the same for all twenty cycles: denaturation at 95"C for 1 minute, primer annealing at 60°C for 1 minute, and amplification for 1.5 minutes at 72'C.
  • the amplification product was gel-purified and digested with Spel.
  • the amplification product was ligated into Stul-Spel cleaved pHIL7SP8 using standard methods.
  • the ligation mixture was used to transform E. coli WK6 selecting for ampicillin resistant clones.
  • a correct insert sequence in one of the resulting plasmid clones, pYAM7SP-AceNI3 was selected to transform the P. pastoris yeast strain GTS115(his4) , as described in Example 12(B) .
  • Selection of His + transformants and subsequent selection for AceNIF3 expression were performed as described in Example 12(B) .
  • the presence of AceNIF3 in Pichia cell supernatant was detected and quantified in a competitive binding assay with biotinylated NIF1 (Example 1) .
  • Pichia cell supernatant was obtained by centrifugation. The crude supernatant was shown to inhibit the adhesion of human neutrophils to plastic.
  • NIF-IFL region coding for the mature protein is fused at its N-terminus to the secretion signal sequence derived from the pelB gene and at its C-terminal end to the filamentous phage M13 gene III (gill) .
  • This gene fusion was placed under the transcriptional control of the Plac promoter.
  • Some of the pelB codons were replaced by synonymous triplets so that the secretion signal contains an Ncol restriction site.
  • An extra Ala-codon was introduced between the pelB and NIF-IFL regions such that the junction matches more closely the prokaryotic prototype signal sequence processing site.
  • the NIF-IFL and pglll (product of gill) encoding regions are separated by (i) a linker sequence in which a Notl site is embedded and (ii) a TAG (amber) triplet which serves as a translational stop codon in a su ' strain but is frequently read as a sense codon in su + bacterial cells.
  • pAN-NIF-lFL A schematic representation of the phagemid vector, designated pAN-NIF-lFL, is shown in Figure 21.
  • pAN-NIF-lFL was constructed by (i) PCR-amplification of the NIF-IFL coding region with primers that contain 5'-extensions whose sequence is such that (ii) the Ncol/Notl directional cloning of the gel-purified PCR fragment in the recipient vector results in the intended in frame fusion of the pelB, NIF-IFL, and gill coding elements.
  • the NIF-IFL coding region was PCR-amplified making use of the following two oligonucleotide primers:
  • LJ046 5'-GTCGCAACTG-CGGCCCAGCC-GGCCATGGCC-GCTAATGAAC-ACAACCTGA G-GTGC (54-mer; 5'-primer; The Ncol site and NIF-IFL N-terminus are underlined) LJ046:
  • NIF-IFL region and flanking sequences present in pAN-NIF-lFL were entirely sequenced to rule out the presence of unwanted mutations.
  • B Display of functional NIF bv filamentous phages.
  • the pAN-NIF-lFL phagemid-vector has the potential to code for a NIF-lFL-pglll fusion protein.
  • a so-called 'helper'-phage this fusion protein can, during morphogenesis, become incorporated into filamentous virions (both 'helper'-phages and pseudo-virions which encapsidate one specific strand of the phagemid) .
  • Phage particles were rescued with M13-VCS 'helper'-phage (Stratagene) infection as follows.
  • a 1 ml culture of TGl[pAN-NIF-lFL] grown at 37'C in 2xTY (2xTY: Tryptone 16 g/L; Yeast extract 10 g/L; NaCl 5 g/L) containing 100 ⁇ g/ml carbenicillin (or ampicillin) and 1% glucose to a density of ODgoo n ⁇ - - 0.5-0.6 is infected with M1-3-VCS at a multiplicity of infection of -20.
  • the infected culture is incubated at 37°C for 30 minutes without shaking and then for another 30 minutes with shaking.
  • a 10 ml prewarmed 2xTY aliquot containing both carbenicillin (100 ⁇ g/ml) and kanamycin (50 ⁇ g/ml) is inoculated with the 1 ml infected culture.
  • the mixture is incubated with shaking first for 60 minutes at 37°C and then overnight at -30°C After removal of the infected cells by centrifugation 1:5 volume 20% polyethyleen glycol/2.5 M NaCl is added to the supernatant.
  • the precipitated phages are collected by centrifugation and resuspended in PBS (Na 2 HP0 4 .2H 2 0 1.14 g/L; KH 2 P0 4 0.2 g/L; NaCl 8.0g/L; KC1 0.2 g/L; pH 7.3) .
  • the rescued phages were shown to display functionally active NIF in several assays: (A) Western blot (After fractionation by SDS-10% PAGE, phage proteins were transferred onto ProBlott (Applied Biosystems Inc. , Foster City, CA) membrane and incubated with rabbit anti-phage serum and goat anti-rabbit alkaline phosphatase conjugate.
  • CDllb/CDl ⁇ -ELISA see Figure 22
  • NIF-phage were then assayed for binding to CDllb/CDl ⁇ (non-displaying phage were used as negative control)
  • CDllb/CD18-coated wells were prepared either by direct immobilization using 0.25 ⁇ g/ml immunopurified CDllb/CDl ⁇ receptor (Diamond et al., 1990, J. Cell Biol., ill, 3129-3139), or by immuno-capture with monoclonal antibody LM2 (ATCC number: HB 204) .
  • Binding of phages was detected with rabbit anti-phage antiserum and goat anti-rabbit alkaline phosphatase conjugate. Binding of phages to the immobilized receptor was shown to occur only when they display the NIF protein. It was also shown that NIF-phage are not able to bind to the LM2 monoclonal antibody nor to the CDllb/CD18-coated wells after a pre-incubation with ImM Pichia-produced recombinant NIF-IFL for 30 minutes) ; (C) 3D2-ELISA (3D2 is a non-neutralizing mouse monoclonal antibody specific for NIF (see Example 26) . In contrast to non-displaying control phages, NIF-phage were found to bind to
  • NIF-phage binding could be eliminated by either blocking the 3D2-wells with 1 mM Pichia-produced recombinant NIF-IFL or blocking the NIF-phages with 1 mM 3D2 monoclonal antibody) ; and (D) Panning against CDllb/CDl ⁇ (pAN-NIF-lFL phage (10 10 virions) were mixed with an equal amount of irrelevant non-displaying phage (fd-tet; 10 10 virions) , diluted in 100 ⁇ l Binding Buffer (PBS, 1 mM CaCl 2 , 1 mM MgCl 2 , 0.4% Tween-20 and 2% Skim-Milk) and incubated in a CDllb/CD18-coated microtiter-well. After incubation for
  • the PCR primers contain 5'-extensions which incorporate restriction sites allowing the facile unidirectional cloning of the amplification product in an appropriate display vector.
  • the phagemid display vector containing the NIF gene, pAN-NIF-lFL is suitable for the production of NIF-IFL in both a phage-attached form and as 'free' soluble protein.
  • the pAN-NIF-lFL phagemid-vector has the potential to direct the synthesis of the NIF-IFL protein in a 'free' (i.e., not phage-attached) form.
  • Cell-free supernatant was filtered (0.2 ⁇ m) and submitted to a diafiltration on a polyethersulfone omega membrane (30kDa cut-off; 0.75 ft 2 ; Filtron) with 10 volumes of 50 mM citric acid pH 3.5 containing ImM EDTA. After adjustment to pH 7.4 by adding 1 M Tris-HCl, the solution was left on ice for at least one hour. Precipitated material was removed by filtration (0.2 ⁇ m) . The cleared supernatant was submitted to a second dialfiltration (10 volumes 10 mM phosphate pH 7.4). Afterwards calcium chloride was added to a final concentration of 0.3 mM.
  • the solution was applied on a MacroPrep (40 ⁇ m) Hydroxyapatite (Bio-Rad Laboratories) column equilibrated with 10 mM phosphate pH 1,4 and containing 0.3 mM CaCl 2 . After washing with 5 column volumes of the equilibration buffer, the recombinant NIF protein was eluted with 90 mM phosphate pH 7.4. Fractions containing recombinant NIF were identified by binding assays on LM2/CDllb/CDl ⁇ plates and pooled.
  • the protein present in the pooled fractions was further purified by reversed phase chromatography on a Poros Rl/H (Perseptive Biosystems) column equilibrated with 10 mM ammonium formate pH 6.4 and 10% acetonitrile. Recombinant NIF was eluted by increasing the acetonitrile concentration. The fractions containing NIF were identified by gel-electrophoresis and were pooled. Acetonitrile present in this pool was removed in a rotavapor before freeze-drying. The dry protein was redissolved in PBS and applied on a Superdex
  • the 5'-primer was composed of two restriction sites (EcoRl and Hpal) and the 23 first nucleotides of the region beginning at the 5'-end of proteolytically processed NIF and the succeeding ⁇ codons.
  • the codon for the first residue of the mature NIF was altered from AAT to AAC (both codons translate to asparagine) and constitutes part of the Hpal restriction sites
  • Amplification was accomplished using 100 pmol of each primer, 2 units of Vent polymerase in IX Vent buffer (New England Biolabs, Beverly, MA), and 0.2 mM of each of dATP, dCTP, dGTP, and dTTP.
  • IX Vent buffer New England Biolabs, Beverly, MA
  • 0.2 mM of each of dATP, dCTP, dGTP, and dTTP One hundred nanograms of Bluescriptll-containing NIF-IFL were used as template DNA.
  • the PCR conditions were the same for all twenty cycles: denaturation at 95°C for 1 minute, primer annealing at 60'C for l minute, and amplification for 1.5 minutes at 72"C.
  • the amplification product was gel-purified and digested with EcoRl and Hindlll.
  • the amplification product was then ligated into EcoRl-Hindlll cleaved pMa5- ⁇ and pMc5-8 respectively
  • NIF-IFL protein contains seven potential N (Asparagine) -glycosylation sites (consensus N-X-T/S amino acid sequence) .
  • pMa5-hNIFl/ ⁇ Gll-5 is a derivative of pMa5-hNIFl (see above) in which five potential N-glycosylation sites of NIF-IFL have been modified by substituting glutamine residues for each of the asparagine residues in the corresponding consensus sequences. These residues are Asn 10 , Asn 18 , Asn 87 , Asn 110 , and Asn 130 , where the number in superscript corresponds to the . mino acid residue number of NIF-IFL (see Fig. 8) .
  • Stepwise site-directed mutagenesis was performed following the methodology described in Stanssens et al., (1989) , Nucl. Acids Res. 12 4441-4454, and using the following oligonucleotides: (I) ⁇ G11:dCCGGGCATTTCGGTACCTTGCTGCGGGCACCTC,
  • the strategy outlined in (B) above was performed in parallel to construct another NIF-IFL derivative, pMa5-NIF-lFL/ ⁇ hG16-7, in which the potential N-glycosylation sites G16 and G17 of NIF-IFL have been modified by substituting glutamine residues for each of the asparagine residues in the corresponding consensus sequences. These residues are Asn 197 , and Asn 223 .
  • the Hpal restriction site (GTTAAC) present in the NIF-IFL coding sequence was removed by introducing a silent mutation at the appropriate position: the AAC codon for Asn 166 was replaced by a AAT codon.
  • Stepwise site-directed mutagenesis was performed following the methodology described in Stanssens et al., (1989), Nucl. Acids Res. 12: 4441-4454, and using the following oligonucleotides:
  • the oligo- nucleotides VII and VIII were annealed together to the ssDNA template to modify the glycosylation site G17 and to remove the Hpal restriction site.
  • a resulting plasmid clone harbouring the two intended sites altered was then used to prepare ssDNA template for the next mutagenesis round in order to modify the G16 remaining site using the appropriate oligonucleotide (VI) .
  • the NIF-IFL derivative hNIFl/ ⁇ h,Gll-7 was constructed using standard methods by combining appropriate fragments prepared from the vectors pMa5-hNIFl/ ⁇ Gll-5 (prepared as in (B) above) and pMa5-NIF-lFL/ ⁇ h,G16-7 (prepared as in (c above) .
  • a one base pair deletion in the NIF sequence (a missing G nucleotide in the Gly 201 GGA codon) revealed by this sequence analysis was corrected by site directed mutagenesis using the oligonucleotide dGTAAATCGGCTGTCCTTCAGTTTTCTG.
  • the segment of DNA encoding hNIFl/ ⁇ Gll-5 was PCR-amplified from a subclone of pMa5-hNIFl/ ⁇ Gll-5 following the methodology described in Example 12(B) and using the same set of primers. After purification, the amplification product was digested with Spel and ligated into Stul-Spel cleaved pHIL7SP8 using standard methods. The ligation mixture was used to transform ______ coli WK6, and ampicillin resistant clones were obtained on ampicillin plates. Based on restriction and DNA sequence analysis, a correct insert sequence in one of the resulting plasmid clones, pYAM7SP-hNIFl/ ⁇ Gll-5, was selected to transform the P. pastoris yeast strain GTS115(his4) , as described in Example 12(B). Selection of His+ transformants and subsequent selection for NIF-1FL/ ⁇ G11-5 expression were performed as described in
  • Example 12 (B) .
  • the Hpal-Spel fragment of DNA encoding hNIFl/ ⁇ h,Gll-7 was prepared from the vector pMa5-hNIFl/ ⁇ h,Gll-7 and ligated into Stul-Spel cleaved pHIL7SP8 using standard methods. The ligation mixture was used to transform I .
  • coli WK6, and ampicillin resistant clones were obtained on ampicillin plates. Based on restriction and DNA sequence analysis, a correct insert sequence in one of the resulting plasmid clones, pYAM7SP-hNIFl/ ⁇ h,Gll-7, was selected to transform the P. pastoris yeast strain GTS115(his4) , as described in Example 12(B) . Selection of His+ transformants and subsequent selection for NIF-lFL/ ⁇ h,Gll-7 expression were performed as-described in Example 12(B). The presence of NIF-lFL/ ⁇ h,Gll-7 in Pichia cell supernatant was detected and quantified using the LM2/CDllb/CDl ⁇ based ELISA with 3D2-HRP detection (see Example 1) .
  • NIF-lFL/ ⁇ h,Gll-7 Recombinant NIF-lFL/ ⁇ h,Gll-7 was purified by hydroxyapatite and reverse-phase chromatography (see Example 23; the gelfiltration step on Superdex was omitted) . Under non-reducing conditions, the purified protein was found to migrate as a single band on SDS-PAGE (4-20% gradient gel; Novex) , with an apparent molecular weight of about 30kDa. The observed higher mobility and apparent lesser heterogeneity of NIF-1FL/ ⁇ G11-5 and NIF-lFL/ ⁇ h,Gll-7 compared to NIF-IFL is likely due to the relatively decreased extent of glycosylation of these mutants compared to the wild-type proein.
  • N-glycosylation sites does not affect the potency of the NIF-IFL molecule as measured in this in vitro assay.
  • mice Female balb-c mice between the ages of 8 to 15 weeks were inoculated subcutaneously with 10 ⁇ g of recombinant NIF (from Example 12) in Complete Freund's Adjuvant, then 3 weeks later were inoculated subcutane- ously with 10 ⁇ g of the recombinant NIF in Incomplete
  • mice producing polyclonal antibody to recombinant NIF were determined by immunoassay of their seruict ' using 125 I labeled recombinant NIF and immobilized goat anti-mouse IgG. Mice having a titer between 1:25,000 to 1:50,000 were selected.
  • Spleen cells were isolated from the mice and were fused with tumor cells in a 5:1 ratio of spleen cells to tumor cells.
  • Hybridoma cells expressing monoclonal antibody binding to NIF were selected by the same immunoassy.
  • Monoclonal antibody was expressed from the selected hybridoma cells by inoculation mterperi ⁇ toneally of 5 x 10° hybridoma cells into pristane-primed female balb-c mice.
  • One monoclonal antibody designated 3D2 was shown to bind both hookworm-derived NIF and Pichia-produced recombinant NIF-IFL by antigen capture assay (E. Harlow and D.P. Lane, Antibodies : A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988) , pp. 192-193).
  • the resin was again collected on a frit, and washed with 10 mM sodium phosphate, 0.15 M sodium chloride, pH 7.3 until the solution flowing through the frit was pH 7.3.
  • the resin was stored 4 ' n lOmM sodium phosphate, 0.15 M sodium chloride, 0.05% sodium azide, pH 7.3, until use. Two millilters of 3D2/Emphaze resin coupled using this procedure was found to bind 1.4 mg of purified NIF protein.
  • a 150 ml Two Step coupling was performed in which 2.25 g of 3D2 antibody in 450 ml of 0.5 M Tris-HCl, pH 4.0 at 4°C was mixed with 19 g of Emphaze Biosupport Medium in a 1000 ml microcarrier spinner flask (Bellco Glass Inc., Vineland, New Jersey) for 10 minutes at 4°C. Forty two grams of sodium sulfate was added to the Emphaze slurry and this was stirred for an additional 10 minutes at 4'C. The pH was raised to 9.0 by dropwise addition of 1 M NaOH through the arms of the spinner flask. The reaction was allowed to proceed for 60 minutes at 4"C with stirring.
  • the Emphaze slurry was collected over a 90 mm, 0.45 micron filter (Corning Glass Works, Corning, New York) , and remaining antibody solution was drained.
  • the resin was washed with 1 liter of 0.01 M sodium phosphate, 0.15 M sodium chloride, pH
  • the reaction was quenched by the addition of 500 ml of 1.0 M ethanolamine, pH 9.3 to the resin. The quenching reaction was allowed to continue 2.5 hours at 4°C with stirring.
  • the resin was again collected over a 90mm, 0.45 micron cellulose acetate filter, and washed with 0.2 M sodium phosphate, 0.5 M sodium chloride, pH 7.3 until the solution flowing through the filter was pH 7.3.
  • the resin was suspended in 0.01 M sodium phosphate, 0.15 M sodium chloride, 0.05% sodium azide, pH 7.3, until use. A 2 ml portion of resin from this coupling bound 1.5 mg of NIF protein, the same amount of NIF protein bound by resin coupled in a 2 ml reaction.
  • the concentrate was filtered through a 90mm, 0.22 micron cellulose acetate filter (Corning). Approximately 150 mg of NIF protein was applied to a 400 ml 3D2/Emphaze column at 20 ml/min. The concentrate was washed from the column with 400 ml of 0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.3 at 20 ml/min. The column flowthrough was collected and retained. The column was then washed with 400 ml of 1 M NaCl and the wash was discarded. The recombinant NIF protein bound to the column was eluted by applying 800 ml of 0.1 M glycine, pH 2.5.
  • the purified NIF protein from the column was brought to neutral pH by the dropwise addition of 1 M Tris base.
  • the column was then re-equilibrated to loading conditions by passing 800 ml of 0.1 M sodium phosphate, 0.15 M sodium chloride, pH
  • NIF protein When approximately 1 g of NIF protein had been purified by the 3D2/Emphaze column, the protein was pooled and then concentrated using an Easyflow 20 kDA polysulfone concentration apparatus (Sartorius) . The concentrated protein was then applied at a flow rate of 10 ml/min. to a 60 cm x 600 cm Superdex* 200 prep gel filtration column (Pharmacia, Piscataway, New Jersey) equilibrated in 0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.3. The only peak observed during elution (870 - 1050 ml) corresponds to NIF protein.
  • Neutrophil Inhibitory Factor is an Inhibitor of Eosinophil Adhesion to Vascular Endothelial Cells
  • NIF was assayed for effect on adhesion of human eosinophils to cytokine-stimulated endothelial cells.
  • Eosinophils were isolated from normal individuals as described by Moser et al (1992a) [J. Immunol. 149:1432-1438] .
  • Isolated eosinophils were cultured in the presence of 10 pM GM-CSF and 10 pM IL-3 for 24 hours following the procedure of Moser et al (1992a) .
  • Endothelial cells were harvested from umbilical cord veins, seeded in tissue culture flasks and transferred to 24-well plates as described by Moser et al (1992a) .
  • the adhesion assay was carried out following the procedure described by Moser et al (1992a) . Briefly, human umbilical vein endothelial cell (HUVEC) monolayers were washed with Hank's balanced salt solution (HBSS) and preincubated with 500 ⁇ l of TNF ⁇ at a final concentration of 10 ng/ml for 4 hours at 37'C. Immediately before use in adhesion assays, HUVEC monolayers were washed. Next, 2.5 X 10 s eosinophils in 500 ⁇ l of HBSS containing 5 mg/ml of purified human albumin were layered onto the washed HUVEC monolayers.
  • HUVEC Hank's balanced salt solution
  • the 24-well plate was submerged three times in a bath of 300 ml PBS to remove loosely adherent eosinophils. Plates were dried at 4°C and the number of adherent neutrophils was quantitated by measuring peroxidase activity, as described in Moser et al, 1992b [Blood 79:2937] .
  • Recombinant NIF inhibited adhesion of GM-CSF/IL-3 primed human neutrophils to TNF-activated HUVEC monolayers, to a maximum of approximately 63% inhibition at 100 nM rNIF. About 50% inhibition of adhesion was obtained in the presence of approximately 10 nM rNIF (see Figure 13).
  • NIF neuropeptide
  • Biotinylated rNIF was prepared as described above for derivatization of NIF purified from hookworm homogenates with the exception that the rNIF was derivatized at a final biotin-LC-hydrazide concentration of 0.14 mM.
  • a buffy coat preparation of human leukocytes, suspended in 2% newborn calf serum, 1% NaN 3 (PBS-NCS) was incubated with biotinylated rNIF (0.6 ⁇ g/ml ) for 5 minutes at room temperature. Red blood cells were lysed by addition of 3 ml 150 mM NH stampC1, 1% NaN 3 for 5 minutes at room temperature.
  • the cells were washed twice in 1 ml PBS-NCS and resuspended in 50 ⁇ l wash buffer (5% newborn calf serum, 0.1% NaN 3 in PBS) containing 0.25 ⁇ g/ml streptavidin-phycoerythrin (Pharmingen) . After 15 minutes at 4 * C the cells were washed and resuspended in 0.5 ml wash buffer.
  • Flow cytometry was performed with a FACScan * instrument (Becton Dickinson) using Lysys II * software (Becton Dickinson) .
  • Leukocyte populations were electronically gated using cytograms of forward versus right angle light scatter. Background binding was determined using biotinylated BSA (Pierce) .
  • Fab' FITC-conjugated goat
  • Caltag streptavidin-phycoerythrin
  • Cys 128 and Glycine 321 residues Cys 128 and Glycine 321
  • the fusion is a 8 amino acid tag peptide termed FLAG * -
  • the PCR primers used to clone I-domain were based on amino acid sequences of the CDllb polypeptide (Gly lll -Ala 318 ) .
  • the primer sequences were 5'-CCA-AAG-CTT-GGA-TCC-AAC-CTA- CGG-CAG-CAG-CC-3' (primer 1) , and 5'-CCA-TCT-AGA-CGC- AAA-GAT-CTT-CTC-CCG-AAG-CT-3' (primer 2).
  • Primer 1 contains an additional 5'-Hindlll restricion endonuclease site
  • primer 2 contains an additional 3 ' -XbaI site.
  • the primers were used pairwise with random-primed single-stranded cDNA (cDNA Synthesis Plus, Amersham) synthesized from human monocyte total RNA (see Example 10 for protocols) .
  • the starting material was -0.5 g human monocytes.
  • PCR conditions were: denaturation at 95 'C for 30 seconds, annealing at 45 °C for 30 seconds, elongation at 72 'C for 2 minutes, for 30 cycles. All PCR reagents were from Perkin-Elmer.
  • PCR amplification produced a fragment containing a -615 base pair I-domain coding region with appropriate 5'- and 3'-restriction sites.
  • the Hindlll/Xbal-digested fragment was ligated into the Hindlll/Xjal-cleaved plasmid vector pFLAG-1 TM (International Biotechnologies, Inc) .
  • the construct was used to transform Epicurean Coli SURE * competent cells (Stratagene) , which were plated on LB media agar plates containing 100 ⁇ g/ml ampicillin and 0.4% glucose for selection. Three milliter overnight cultures inoculated with transformed colonies were grown at 37 ⁇ in LB + 100 ⁇ g/ml ampicillin. Isolation of plasmid DNA from the overnight cultures was performed with a Magic Miniprep kit
  • the 500 ml culture was grown at 37 °C to an ODj- o of 0.400 (-120 minutes), induced with 1.7 mM isopropyl b-D-thiogalactopyranoside (Sigma), and grown for two additional hours at 37 °C Cells were harvested by centrifugation at 5,000 g for 5 minutes, 25 °C. The cells were then resuspended in 50 ml of an extraction buffer-1 (50 mM Tris, pH 8.0, 5 mM EDTA, 0.25 mg/ml lysozyme, and 50 ⁇ g/ml NaN 3 ) , and allowed to incubate for 5 minutes at 25 °C .
  • an extraction buffer-1 50 mM Tris, pH 8.0, 5 mM EDTA, 0.25 mg/ml lysozyme, and 50 ⁇ g/ml NaN 3
  • extraction buffer-2 1.5 M NaCl, 100 mM CaCl 2 , 100 mM MgCl 2 , 0.02 mg/ml DNase 1, and 50 ⁇ g/ml ovomucoid protease inhibitor
  • this suspension was allowed to incubate for 5 minutes at 25 "C.
  • Cells were further lysed by sonication for 4 cycles of 30 seconds at 70 W on a Branson Sonic power sonifier. Insoluble cellular debris was separated by centrifugation at 25,000 g for 60 minutes at 4 * C
  • the cell lysate containing CDllb I-domain fusion peptide was stored at -70 ' C .
  • Monoclonal antibody/CDllb I-domain fusion protein complexes were formed by incubating the cell lysate prepared as described above with a monoclonal antibody that recognized the FLAG peptide of the CDllb I-domain fusion protein (anti-FLAG Ml; International Biotechnologies, Inc.). These complexes were incubated with ,25 I-rNIF and precipitated with protein A-sepharose (Calbiochem) . One microgram of Ml monoclonal antibody was incubated with 390 ⁇ l of the cell lysate that contained CDllb I-domain fusion peptide, in the presence of 10 s cpm 125 I-rNIF (specific activity 47.5 ⁇ Ci/ ⁇ g; see Example 14(B)).
  • the total reaction volume was 400 ⁇ l, and the reaction proceeded for 18 hours at 4'C.
  • Immune complexes were precipitated with 100 ⁇ l protein A-Sepharose (Pharmacia) .
  • the protein A-Sepharose was prepared as a 1:1 slurry in TACTS 20 buffer and preblocked with 0.5% BSA. After precipitation, Sepharose beads were washed three times with TACTS 20 buffer, and immune complexes were released by the addition of Tris-glycine sample buffer (Novex) under reducing conditions (100 mM dithiothreitol) . Precipitated, labeled proteins were resolved by 4-20% gradient SDS-PAGE (Novex) and visualized by autoradiography.
  • the 125 I-rNIF was precipitated by Ml monoclonal antibody in the presence of cell lysate that contained recombinant I-domain peptide.
  • I23 I-rNIF was not precipitated in the absence of this cell lysate or when the Ml monoclonal antibody was substituted with a monoclonal antibody that did not react with the the FLAG portion of the fusion protein (anti-gpllbllla) .
  • Ml did not precipitate I2S I-rNIF in the presence of another fusion protein comprising FLAG and bacterial alkaline phosphatase (pFLAG-BAP; International Biotechnologies, Inc.).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Toxicology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

Compositions enrichies destinées au facteur inhibiteur des neutrophiles, lequel inhibe l'activité neutrophile y compris l'adhésion sur des cellules endothéliales vasculaires. L'invention concerne également des facteurs inhibiteurs des neutrophiles qui sont produits par recombinaison et qui inhibent également l'activité neutrophile. Ces compositions peuvent comprendre une glycoprotéine isolée provenant de nématodes. Lesdites compositions et les facteurs inhibiteurs des neutrophiles produits par recombinaison sont utiles dans la thérapie de pathologies entraînant des réponses inflammatoires anormales ou indésirables.
PCT/US1993/012626 1992-05-11 1993-12-23 Nouveaux inhibiteurs de neutrophiles WO1994014973A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP94907114A EP0682714A4 (fr) 1992-12-24 1993-12-23 Nouveaux inhibiteurs de neutrophiles.
JP6515483A JPH08505055A (ja) 1992-12-24 1993-12-23 新規好中球抑制剤
AU60805/94A AU694103B2 (en) 1992-05-11 1993-12-23 Novel neutrophil inhibitors

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US99697292A 1992-12-24 1992-12-24
US07/996,972 1992-12-24
US08/060,433 US6756211B1 (en) 1992-05-11 1993-05-11 Neutropohil inhibitors
US08/060,433 1993-05-11
US08/151,064 US6962795B1 (en) 1992-05-11 1993-11-10 Neutrophil inhibitors
US08/151,064 1993-11-10

Publications (1)

Publication Number Publication Date
WO1994014973A1 true WO1994014973A1 (fr) 1994-07-07

Family

ID=27369840

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/012626 WO1994014973A1 (fr) 1992-05-11 1993-12-23 Nouveaux inhibiteurs de neutrophiles

Country Status (6)

Country Link
EP (1) EP0682714A4 (fr)
JP (1) JPH08505055A (fr)
AU (1) AU694103B2 (fr)
CA (1) CA2152599A1 (fr)
NZ (2) NZ261662A (fr)
WO (1) WO1994014973A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115057915A (zh) * 2021-12-28 2022-09-16 四川农业大学 一种大肠杆菌表达蛋白Cry5B的制备及抗大熊猫蛔虫活性

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4524816B2 (ja) * 1998-07-06 2010-08-18 東ソー株式会社 Ｉｌ−６レセプター・ｉｌ−６直結融合蛋白質
CN101245110B (zh) * 2007-02-16 2010-09-15 鲁南制药集团股份有限公司 重组中性粒细胞抑制因子和水蛭原嵌合蛋白及其药物组合物

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4338305A (en) * 1975-03-24 1982-07-06 American Home Products Corporation Use of LRH and LRH agonists
US4591552A (en) * 1982-09-29 1986-05-27 New York Blood Center, Inc. Detection of hepatitis B surface antigen (or antibody to same) with labeled synthetic peptide
US5082778A (en) * 1986-06-03 1992-01-21 Unilever Patent Holdings B.V. Production of guar alpha-galactosidase by hosts transformed with recombinant dna. methods

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4338305A (en) * 1975-03-24 1982-07-06 American Home Products Corporation Use of LRH and LRH agonists
US4591552A (en) * 1982-09-29 1986-05-27 New York Blood Center, Inc. Detection of hepatitis B surface antigen (or antibody to same) with labeled synthetic peptide
US5082778A (en) * 1986-06-03 1992-01-21 Unilever Patent Holdings B.V. Production of guar alpha-galactosidase by hosts transformed with recombinant dna. methods

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Immun. Rev., Number 114, issued 1990, CARLOS et al., "Membrane Proteins Involved in Phagocyte Adherence to Endothelium", pages 5-28, see the entire document. *
Immunol. Lett., Volume 16, issued 1987, SOULSBY, "The Evasion of the Immune Response and Immunological Unresponsiveness: Parasitic Helminth Infections", pages 315-320, see entire document. *
Infect. Immun., Volume 48, Number 1, issued May 1985, GASBARRE et al., "Suppression of Antigen and Mitogen- Induced Proliferation of Bovine Lymphocytes by Excretory -Secretory Products of Oesophagostomum Radiatum", pages 540-545, see entire document. *
Int. J. Parasit., Volume 19, Number 1, issued 1989, MONROY et al., "Low Molecular Weight Immunosuppressors Secreted by Adult Nematospiroides Dubius", pages 125-127, see entire document. *
Mol. Biochem. Parasit., Volume 41, issued 1990, SAVIN et al., "Characterization, Cloning, and Host-Protective Activity of a 30-Kilodalton Glycoprotein Secreted by the Parasitic Stages of Trichostronglyus Colubrifomis", pages 167-176, see entire document. *
Mol. Biochem. Parasit., Volume 44, SHEPARD et al., "A Protein Secreted in Vivo by Echinococcus Granulosus Inhibits Elastase Activity and Neutrophil Chemotaxis", pages 81-90, see entire document. *
See also references of EP0682714A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115057915A (zh) * 2021-12-28 2022-09-16 四川农业大学 一种大肠杆菌表达蛋白Cry5B的制备及抗大熊猫蛔虫活性
CN115057915B (zh) * 2021-12-28 2024-04-26 四川农业大学 一种大肠杆菌表达蛋白Cry5B在制备抗大熊猫蛔虫成虫和L4期幼虫活性药物中的应用

Also Published As

Publication number Publication date
EP0682714A4 (fr) 1997-06-11
CA2152599A1 (fr) 1994-07-07
NZ261662A (en) 1997-03-24
JPH08505055A (ja) 1996-06-04
AU6080594A (en) 1994-07-19
EP0682714A1 (fr) 1995-11-22
NZ314241A (en) 1999-01-28
AU694103B2 (en) 1998-07-16

Similar Documents

Publication Publication Date Title
US5747296A (en) Method of detecting neutophil inhibitory factor mimics
Kaumeyer et al. The mRNA for a proteinase inhibitor related to the HI-30 domain of inter-α-trypsin inhibitor also encodes α-1-microgiobulin (protein HC
Ando et al. Molecular cloning, sequencing, and characterization of cDNA for sarcotoxin IIA, an inducible antibacterial protein of Sarcophaga peregrina (flesh fly)
US5789178A (en) Neutrophil inhibitors
US5521296A (en) Glycoprotein and gene coding therefor
JPH05501398A (ja) 組換え産生したヒト細胞膜補助因子タンパク（ｍｃｐ）
EP0731709B1 (fr) Des inhibiteurs de neutrophiles provenant des nematodes
AU694103B2 (en) Novel neutrophil inhibitors
EP0550506B1 (fr) Nouveau facteur chimiotactique
AU589598B2 (en) Brain derivable polypeptide factors and antibodies
US5708141A (en) Neutrophil inhibitors
US4714683A (en) Polypeptide tumor inhibitors and antibodies thereto
EP0728141B1 (fr) Procede d'identification de composes antiparasitaires
NZ516533A (en) Use of neutrophil inhibitors in the manufacture of medicaments to inhibit inflammatory responses and neutrophil activity
NZ276226A (en) Gpd protein, antimalaria vaccine, dna coding for the protein
AU2005318173B2 (en) CC-chemokine antagonists
US4963485A (en) Brain derivable polypeptide factors and antibodies thereto
US6036958A (en) Tsetse thrombin inhibitor
US5786451A (en) 23 Kd human retinal CAR antigen
EP1311283A2 (fr) Omega-conopeptides
AU697868C (en) The cloning of Duffy blood group antigen
AU2005246980A1 (en) Gamma-carboxyglutamate containing conopeptides

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA FI JP KR NO NZ

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 261662

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 2152599

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1994907114

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1994907114

Country of ref document: EP